Directed evolution of a regioselective halogenase for increased thermostability

ABSTRACT

Compounds and methods are providing involving RebH variants with improved properties. directed evolution based on random mutagenesis was employed to generate a series of RebH variants. RebH variants with improved thermostability and increased activity at elevated temperatures were generated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371of International Application No. PCT/US2014/067661, filed Nov. 26, 2014,which claims priority to U.S. Provisional Application No. 61/909,951,filed on Nov. 27, 2013. The entire contents of each of theabove-referenced disclosures are specifically incorporated herein byreference without disclaimer.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number5R00GM087551-03 awarded by The National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the fields of molecular biology,biochemistry, organic chemistry and optical biophysics. Moreparticularly, it concerns the generation of RebH mutants and analysisthereof for the halogenation of organic compounds and thermal stability.

DESCRIPTION OF RELATED ART

Halogenated organic compounds are extensively employed as buildingblocks, synthetic intermediates, and end use products for industrial,materials, agrochemical, and pharmaceutical applications due to theirunique reactivity and physical properties. A multitude of methods forthe halogenation of alkyl and alkenyl compounds have been described.These include Kharasch metal-catalyzed free radical addition of CXCl₃compounds to alkenes, epoxide ring-opening by halide nucleophiles, Appelconversion of an alcohol to a halogen, the Hunsdiecker reaction toconvert a carboxylic acid to a chain-shortened halide,Hell-Volhard-Zelinsky halogenation to alpha-halogenate carboxylic acids,and direct halogen addition to alkenes, among others.

Halogenated arenes comprise a particularly important class of compounds.To date, more than 3,800 halogenated natural products have beenidentified, several of which include halogenated arene functionalgroups. The therapeutic natural products Vancomycin, an importantantibiotic isolated from soil fungi, Griseofulvin, an orallyadministered antifungal agent, chlorotetracycline, an antibiotic, andMaytansine, a potent antitumor agent include at least one halogenatedarene in their structures.

Conventional approaches to arene halogenation via electrophilic aromaticsubstitution require harsh chemical oxidants and often suffer from poorregioselectivity. The two primary procedures for the direct halogenationof arenes proceed through relatively harsh conditions. The Sandmeyerreaction employs nitrous acid to convert an aniline to an aryl halidethrough a diazonium salt intermediate. Friedel-Crafts halogenationemploys a metal halide, e.g. FeCl₃ or AlCl₃, to effect the halogenationof an arene. The relatively harsh conditions of the Sandmeyer andFriedel Crafts aromatic halogenation reactions are incompatible withcertain functional groups. Despite the multitude of organic chemicaltransformations available for the halogenation of alkyl and alkenylcompounds, a method for the halogenation of arenes under mild conditionshas yet to be identified.

SUMMARY

Embodiments provided herein are based on the development andcharacterization of RebH mutants that halogenate aromatic compounds. Ina first embodiment, methods and compositions are provided for thebiosynthetic halogenation of arenes. In a further embodiment,compositions comprising mutated variants of RebH demonstrate improvedarene-halogenating activity. In some embodiments mutated variants ofRebH exhibit increased thermal stability over wild-type RebH.

In some embodiments, there are mutated variants of the halogenase RebH.In some embodiments, the mutated variants of RebH are isolatedpolypeptides. In specific embodiments, a RebH variant comprises one ormore amino acid substitutions selected from the group consisting of S2P;I52T; A58V; M71V, M71T, M71A, or M71C; N75K; E96V; D101G; S110P; S110L;F111L; F111S; G112S; G112D; L113D; L113N; L114P; S130L; K145M; K145R;N166S; F171I; K187R; D203A or D203G; T213A; V225I; K237E; V256I; T258A;D264G; T283A; L289P; F312L; T322I; T348A; L380F; T394M; F396L; F396Y;R400C; T413A; E423D; A442V; S448P; L453P; Y455W; F458S; F458L; E461G;F465L; F465C; N467T; N470S; A476T; A476V; V481A; Q494R; T496R; T496A;G504S; and R509Q. In particular embodiments, a RebH variant comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or39 of such amino acid substitutions (and any range derivable therein).SEQ ID NO:1 is the amino acid sequence of a RebH polypeptide. SEQ IDNO:2 is the underlying nucleic acid sequence for the RebH polypeptide.

In some embodiments, a RebH variant polypeptide of SEQ ID NO: 1comprises at least one amino acid substitution, wherein the at least oneamino acid substitution results in improved halogenating activity. Inparticular embodiments, a RebH variant polypeptide is an isolated RebHvariant polypeptide. In some embodiments, a RebH mutant undergoes one ormore subsequent rounds of optimization. Subsequent optimization mayimpart a primary mutation, i.e., a mutation of a wild type amino acidand/or a secondary mutation, i.e., a mutation of a previously mutatedamino acid. The relative importance of the mutation is not reflected bythe use of the term “secondary.” Rather, “secondary” refers to themutation process where a wild type amino acid is mutated in a firstmutating round, then is further mutated to a secondary, different aminoacid in a subsequent optimization process. For example, a RebH variantcomprising a S110P mutation may be further mutated to comprise thesecondary mutation P110L. The effective mutation from WT RebH is S110L.In some embodiments, secondary mutations include P110L and L111S.Subsequent optimization of a RebH mutant may impart secondary, or higherorder mutations, for example, tertiary and quaternary mutations. It isspecifically contemplated that substitutions need not be created in astep-wise fashion but in some embodiments the method of creating theminvolves serial substitutions at the same position.

In particular embodiments, a RebH variant polypeptide comprises one ormore of the following substitutions as compared to SEQ ID NO:1: S2P,I52T, A58V, M71V, M71T, M71A, M71C, N75K, E96V, D101G, S110P, S110L,F111L, F111S, G112S, G112D, L113D, L113N, L114P, S130L, K145M, K145R,N166S, F171I, K187R, D203A or D203G, T213A, V225I, K237E, V256I, T258A,D264G, T283A, L289P, F312L, T322I, T348A, L380F, T394M, F396Y, F396L,R400C, T413A, E423D, A442V, S448P, L453P, Y455W, F458S, F458L, E461G,F465C, F465L, N467T, N470S, A476T, A476V, V481A, Q494R, T496R, T496A,G504S, and R509Q. In particular embodiments, a RebH variant comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or39 of such amino acid substitutions (and any range derivable therein).It is specifically contemplated that variations may be made in RebHpolypeptides whose sequence differs from SEQ ID NO:1. A RebH polypeptidewhose sequence differs from SEQ ID NO:1, for example, a polypeptidesequence that is less than 100% identical but at least 60% identical,may have any of the following amino acids substituted or combinations ofamino acids substituted: the serine corresponding to S2 in SEQ ID NO:1;the methionine corresponding to M71 in SEQ ID NO:1; the asparaginecorresponding to N75 in SEQ ID NO:1; the glutamic acid corresponding toE96 in SEQ ID NO:1; the aspartic acid corresponding to D101 in SEQ IDNO:1; the glycine corresponding to G112 in SEQ ID NO:1; the leucinecorresponding to L114 in SEQ ID NO:1; the serine corresponding to S130in SEQ ID NO:1; the lysine corresponding to K145 in SEQ ID NO:1; theasparagine corresponding to N166 in SEQ ID NO:1; the phenylalaninecorresponding to F171 in SEQ ID NO:1; the aspartic acid corresponding toD203 in SEQ ID NO:1; the threonine corresponding to T213 in SEQ ID NO:1;the valine corresponding to V225 in SEQ ID NO:1; the lysinecorresponding to K237 in SEQ ID NO:1; the valine corresponding to V256in SEQ ID NO:1; the threonine corresponding to T258 in SEQ ID NO:1; theaspartic acid corresponding to D264 in SEQ ID NO:1; the threoninecorresponding to T283 in SEQ ID NO:1; the leucine corresponding to L289in SEQ ID NO:1; the phenylalanine corresponding to F312 in SEQ ID NO:1;the threonine corresponding to T348 in SEQ ID NO:1; the leucinecorresponding to L380 in SEQ ID NO:1; the threonine corresponding toT394 in SEQ ID NO:1; the phenylalanine corresponding to F396 in SEQ IDNO:1; the arginine corresponding to R400 in SEQ ID NO:1; the threoninecorresponding to T413 in SEQ ID NO:1; the glutamic acid corresponding toE423 in SEQ ID NO:1; the leucine corresponding to L453 in SEQ ID NO:1;the phenylalanine corresponding to F458 in SEQ ID NO:1; the glutamicacid corresponding to E461 in SEQ ID NO:1; the phenylalaninecorresponding to F465 in SEQ ID NO:1; the asparagine corresponding toN467 in SEQ ID NO:1; the asparagine corresponding to N470 in SEQ IDNO:1; the alanine corresponding to A476 in SEQ ID NO:1; the glutaminecorresponding to Q494 in SEQ ID NO:1; the threonine corresponding toT496 in SEQ ID NO:1; and/or, the glycine corresponding to G504 in SEQ IDNO:1. These substitutions may be with any amino acid or the amino acidindicated for that position in the following list: S2P, I52T, A58V,M71V, M71T, M71A, M71C, N75K, E96V, D101G, S110P, S110L, F111L, F111S,G112S, G112D, L113D, L113N, L114P, S130L, K145M, K145R, N166S, F171I,K187R, D203A or D203G, T213A, V225I, K237E, V256I, T258A, D264G, T283A,L289P, F312L, T322I, T348A, L380F, T394M, F396Y, F396L, R400C, T413A,E423D, A442V, S448P, L453P, Y455W, F458S, F458L, E461G, F465C, F465L,N467T, N470S, A476T, A476V, V481A, Q494R, T496R, T496A, G504S, andR509Q. In a particular embodiment, a RebH variant comprises the aminoacid substitutions S2P, M71V, K145M, N467T, N470S, and G112S.

In some embodiments, a RebH variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore serine substitutions (and any range derivable therein), which mayor may not be at positions 1 and/or 130 in SEQ ID NO:1. In someembodiments, a RebH variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moremethionine substitutions, which may or may not be at position 71 in SEQID NO:1. In some embodiments, a RebH variant has 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more glutamic acid substitutions (and any range derivabletherein), which may or may not be at positions 96, 423, and/or 461 inSEQ ID NO:1. In some embodiments, a RebH variant has 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more aspartic acid substitutions (and any range derivabletherein), which may or may not be at positions 101, 203, and/or 264 inSEQ ID NO:1. In some embodiments, a RebH variant has 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more glycine substitutions (and any range derivabletherein), which may or may not be at positions 112 and/or 504 in SEQ IDNO:1. In some embodiments, a RebH variant has 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more lysine substitutions (and any range derivable therein), whichmay or may not be at positions 145 and/or 237 in SEQ ID NO:1. In someembodiments, a RebH variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreasparagine substitutions (and any range derivable therein), which may ormay not be at positions 166, 467, and/or 470 in SEQ ID NO:1. In someembodiments, a RebH variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephenylalanine substitutions (and any range derivable therein), which mayor may not be at positions 171, 312, 396, 458, and/or 465 in SEQ IDNO:1. In some embodiments, a RebH variant has 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more threonine substitutions (and any range derivable therein),which may or may not be at positions 213, 258, 283, 348, 394, 413,and/or 496 in SEQ ID NO:1. In some embodiments, a RebH variant has 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more valine substitution (and any rangederivable therein), which may or may not be at positions 225 and/or 256in SEQ ID NO:1. In some embodiments, a RebH variant has 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more leucine substitutions (and any range derivabletherein), which may or may not be at positions 289, 380, and/or 453 inSEQ ID NO:1. In some embodiments, a RebH variant has 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more arginine substitutions (and any range derivabletherein), which may or may not be at position 400 in SEQ ID NO:1. Insome embodiments, a RebH variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore alanine substitutions, which may or may not be at position 476 inSEQ ID NO:1. In some embodiments, a RebH variant has 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more glutamine substitutions (and any range derivabletherein), which may or may not be at position 494 in SEQ ID NO:1.

In some embodiments, a RebH variant polypeptide comprises at least oneamino acid substitution at position 2, 52, 58, 71, 75, 96, 101, 110,111, 112, 113, 114, 130, 145, 166, 171, 187, 203, 213, 225, 237, 256,258, 264, 283, 289, 312, 322, 348, 380, 394, 396, 400, 413, 423, 442,448, 453, 455, 458, 461, 465, 467, 470, 476, 481, 494, 496, 504 and/or509 in SEQ ID NO:1. In further embodiments, a RebH variant polypeptidecomprises at least 3 amino acid substitutions at positions 2, 52, 58,71, 75, 96, 101, 110, 111, 112, 113, 114, 130, 145, 166, 171, 187, 203,213, 225, 237, 256, 258, 264, 283, 289, 312, 322, 348, 380, 394, 396,400, 413, 423, 442, 448, 453, 455, 458, 461, 465, 467, 470, 476, 481,494, 496, 504 and/or 509 in SEQ ID NO: 1. In other embodiments, a RebHvariant polypeptide comprises at least 5 amino acid substitutions atpositions 2, 52, 58, 71, 75, 96, 101, 110, 111, 112, 113, 114, 130, 145,166, 171, 187, 203, 213, 225, 237, 256, 258, 264, 283, 289, 312, 322,348, 380, 394, 396, 400, 413, 423, 442, 448, 453, 455, 458, 461, 465,467, 470, 476, 481, 494, 496, 504 and/or 509 in SEQ ID NO: 1. Inparticular embodiments, a RebH variant comprises or comprises at leastor at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37 or 38 amino acid substitutions (and any range derivable therein)at position 2, 71, 75, 96, 101, 112, 114, 130, 145, 166, 171, 203, 213,225, 237, 256, 258, 264, 283, 289, 312, 348, 380, 394, 396, 400, 413,423, 453, 458, 461, 465, 467, 470, 476, 494, 496, and/or 504 in SEQ IDNO: 1.

In additional embodiments, a RebH variant polypeptide comprises at leastone amino acid substitution at position 2, 52, 58, 71, 75, 96, 101, 110,111, 112, 113, 114, 130, 145, 166, 171, 187, 203, 213, 225, 237, 256,258, 264, 283, 289, 312, 322, 348, 380, 394, 396, 400, 413, 423, 442,448, 453, 455, 458, 461, 465, 467, 470, 476, 481, 494, 496, 504 and/or509 in SEQ ID NO:1, wherein the RebH variant polypeptide is at least 60%identical to SEQ ID NO:1. In additional embodiments, a RebH variantpolypeptide comprises at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, or 37 amino acid substitution(s) atposition 2, 52, 58, 71, 75, 96, 101, 110, 111, 112, 113, 114, 130, 145,166, 171, 187, 203, 213, 225, 237, 256, 258, 264, 283, 289, 312, 322,348, 380, 394, 396, 400, 413, 423, 442, 448, 453, 455, 458, 461, 465,467, 470, 476, 481, 494, 496, 504 and/or 509 in SEQ ID NO:1, wherein theRebH variant polypeptide is at least or at most 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:1 (or any rangederivable therein). It is specifically contemplated that there may besubstitutions at other positions in SEQ ID NO:1. Additionally, a versionof RebH may be used such that it is missing at most or at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more total orcontiguous amino acids (or any range derivable therein) from SEQ IDNO:1.

In some embodiments, a RebH variant polypeptide can halogenate aromaticsubstrates. In some embodiments, a RebF reductase is employed with aRebH variant polypeptide. In particular embodiments, a RebH variantpolypeptide regioselectively halogenates aromatic substrates. In furtherembodiments, a RebH variant polypeptide halogenates aromatic substratesto create novel compounds. In further embodiments, a RebH variantpolypeptide halogenates aromatic substrates in the presence offunctional groups not amenable to existing aromatic chlorinationmethods. In particular embodiments, a RebH polypeptide variant is usedto append halogen isotopes to organic compounds. In further embodiments,a RebH polypeptide variant is used to isotopically label organiccompounds. In some embodiments, a RebH variant polypeptide displaysimproved thermostability over wild-type RebH. In other embodiments, aRebH variant polypeptide displays increased halogenating activity at anelevated temperature. In some embodiments, a RebH variant polypeptide'sincreased halogenating activity includes higher product conversionand/or increased reaction kinetics over wild-type RebH. In yet otherembodiments, a RebH variant polypeptide displays improved catalyticefficiency over wild-type RebH. In further embodiments, a RebH variantpolypeptide halogenates with higher substrate conversion than existingaromatic chlorination methods.

In some embodiments, a RebH variant polypeptide halogenates arylcompounds. In particular embodiments, a RebH variant polypeptidehalogenates the wild-type RebH native substrate tryptophan. In otherembodiments, a RebH variant polypeptide halogenates non-nativesubstrates. In yet other embodiments, a RebH variant polypeptidehalogenates indole, tryptoline, and 2-methyltryptamine. In particularembodiments, a RebH variant polypeptide halogenates using a halideselected from the group consisting of fluoride, chloride, bromide andiodide. In some embodiments, halogenating conditions comprise a mixtureof HEPES, lysate, NaCl, NAD, FAD, glucose, RebF, glucose dehydrogenaseand RebH or a RebH variant polypeptide.

In some embodiments, a RebH variant polypeptide displays prolongedcatalyst lifetime over wild-type RebH. In some embodiments, a RebHvariant polypeptide displays increased tolerance to proteolysis overwild-type RebH. In other embodiments, a RebH variant polypeptidedisplays increased tolerance to organic solvents over wild-type RebH. Insome embodiments, the halogenation reaction proceeds in the absence of aharsh chemical oxidant, for example aluminum chloride, iron (III)chloride or nitrous acid.

In any of the embodiments involving RebH variants, it is contemplatedthat a RebH variant is one that has less than 100% amino acid identityto SEQ ID NO:1. In different embodiments, and in the context of anyother RebH embodiment discussed herein, the RebH variant has, has atleast, or has at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5,99.6, 99.7, or 99.8 percent identity (or any range derivable therein) toSEQ ID NO:1. Additionally or alternatively, RebH variants may have orhave at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470,480, 490, 500, or 510 contiguous amino acids (and any range derivabletherein) from SEQ ID NO:1. In certain embodiments, there are or are atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100 different regions of contiguousamino acids (and any range derivable therein) in a RebH variant thathave, have at most, or have at least 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100 contiguous amino acids from SEQID NO:1 (and any range derivable therein).

In some embodiments, a process for halogenating an aromatic substratecomprises employing a RebH variant polypeptide. In other embodiments, aprocess for halogenating an aromatic substrate comprises mixing anisolated RebH variant polypeptide with the aromatic substrate and atleast one halide source under halogenating conditions. In someembodiments, a RebH variant polypeptide displays at least 98% sequencehomology to SEQ ID NO: 1.

In some embodiments, a RebH variant polypeptide is used in a method toproduce a library of halogenated aromatic compounds. In otherembodiments, a method for producing a library of halogenated aromaticcompounds comprises reacting a plurality of different aromatic compoundswith an isolated RebH variant polypeptide and at least one halide sourceunder halogenating conditions. In particular embodiments, the isolatedRebH variant polypeptide has at least 98% sequence homology to SEQ IDNO: 1. In some embodiments, the isolated RebH variant polypeptidecomprises at least one amino acid substitution at position 2, 52, 58,71, 75, 96, 101, 110, 111, 112, 113, 114, 130, 145, 166, 171, 187, 203,213, 225, 237, 256, 258, 264, 283, 289, 312, 322, 348, 380, 394, 396,400, 413, 423, 442, 448, 453, 455, 458, 461, 465, 467, 470, 476, 481,494, 496, 504 and/or 509 in SEQ ID NO:1.

In some embodiments, the RebH variant comprises any combination ofsubstitutions of one or more amino acids in SEQ ID NO. 1, selected fromthe group consisting of: S2P, I52T, A58V, M71V, M71T, M71A, M71C, N75K,E96V, D101G, S110P, S110L, F111L, F111S, G112S, G112D, L113D, L113N,L114P, S130L, K145M, K145R, N166S, F171I, K187R, D203A or D203G, T213A,V225I, K237E, V256I, T258A, D264G, T283A, L289P, F312L, T322I, T348A,L380F, T394M, F396Y, F396L, R400C, T413A, E423D, A442V, S448P, L453P,Y455W, F458S, F458L, E461G, F465C, F465L, N467T, N470S, A476T, A476V,V481A, Q494R, T496R, T496A, G504S, and R509Q.

In some embodiments, the RebH variant comprises a combination ofsubstitutions in SEQ ID NO: 1 selected from the following combinationsof substitutions (separated by a semicolon): S2P, M71V; S2P, M71T; S2P,M71A; S2P, M71C; S2P, N75K; S2P, E96V; S2P, D101G; S2P, G112S; S2P,L114P; S2P, S130L; S2P, K145M; S2P, N166S; S2P, F171I; S2P, D203A; S2P,D203G; S2P, T213A; S2P, V225I; S2P, K237E; S2P, V256I; S2P, T258A; S2P,D264G; S2P, T283A; S2P, L289P; S2P, F312L; S2P, T348A; S2P, L380F; S2P,T394M; S2P, F396Y; S2P, R400C; S2P, T413A; S2P, E423D; S2P, L453P; S2P,F458S; S2P, E461G; S2P, F465C; S2P, N467T; S2P, N470S; S2P, A476T; S2P,Q494R; S2P, T496R; S2P, G504S; M71V, N75K; M71V, E96V; M71V, D101G;M71V, G112S; M71V, L114P; M71V, S130L; M71V, K145M; M71V, N166S; M71V,F171I; M71V, D203A; M71V, D203G; M71V, T213A; M71V, V225I; M71V, K237E;M71V, V256I; M71V, T258A; M71V, D264G; M71V, T283A; M71V, L289P; M71V,F312L; M71V, T348A; M71V, L380F; M71V, T394M; M71V, F396Y; M71V, R400C;M71V, T413A; M71V, E423D; M71V, L453P; M71V, F458S; M71V, E461G; M71V,F465C; M71V, N467T; M71V, N470S; M71V, A476T; M71V, Q494R; M71V, T496R;M71V, G504S; M71T, N75K; M71T, E96V; M71T, D101G; M71T, G112S; M71T,L114P; M71T, S130L; M71T, K145M; M71T, N166S; M71T, F171I; M71T, D203A;M71T, D203G; M71T, T213A; M71T, V225I; M71T, K237E; M71T, V256I; M71T,T258A; M71T, D264G; M71T, T283A; M71T, L289P; M71T, F312L; M71T, T348A;M71T, L380F; M71T, T394M; M71T, F396Y; M71T, R400C; M71T, T413A; M71T,E423D; M71T, L453P; M71T, F458S; M71T, E461G; M71T, F465C; M71T, N467T;M71T, N470S; M71T, A476T; M71T, Q494R; M71T, T496R; M71T, G504S; M71A,N75K; M71A, E96V; M71A, D101G; M71A, G112S; M71A, L114P; M71A, S130L;M71A, K145M; M71A, N166S; M71A, F171I; M71A, D203A; M71A, D203G; M71A,T213A; M71A, V225I; M71A, K237E; M71A, V256I; M71A, T258A; M71A, D264G;M71A, T283A; M71A, L289P; M71A, F312L; M71A, T348A; M71A, L380F; M71A,T394M; M71A, F396Y; M71A, R400C; M71A, T413A; M71A, E423D; M71A, L453P;M71A, F458S; M71A, E461G; M71A, F465C; M71A, N467T; M71A, N470S; M71A,A476T; M71A, Q494R; M71A, T496R; M71A, G504S; M71C, N75K; M71C, E96V;M71C, D101G; M71C, G112S; M71C, L114P; M71C, S130L; M71C, K145M; M71C,N166S; M71C, F171I; M71C, D203A; M71C, D203G; M71C, T213A; M71C, V225I;M71C, K237E; M71C, V256I; M71C, T258A; M71C, D264G; M71C, T283A; M71C,L289P; M71C, F312L; M71C, T348A; M71C, L380F; M71C, T394M; M71C, F396Y;M71C, R400C; M71C, T413A; M71C, E423D; M71C, L453P; M71C, F458S; M71C,E461G; M71C, F465C; M71C, N467T; M71C, N470S; M71C, A476T; M71C, Q494R;M71C, T496R; M71C, G504S; N75K, E96V; N75K, D101G; N75K, G112S; N75K,L114P; N75K, S130L; N75K, K145M; N75K, N166S; N75K, F171I; N75K, D203A;N75K, D203G; N75K, T213A; N75K, V225I; N75K, K237E; N75K, V256I; N75K,T258A; N75K, D264G; N75K, T283A; N75K, L289P; N75K, F312L; N75K, T348A;N75K, L380F; N75K, T394M; N75K, F396Y; N75K, R400C; N75K, T413A; N75K,E423D; N75K, L453P; N75K, F458S; N75K, E461G; N75K, F465C; N75K, N467T;N75K, N470S; N75K, A476T; N75K, Q494R; N75K, T496R; N75K, G504S; E96V,D101G; E96V, G112S; E96V, L114P; E96V, S130L; E96V, K145M; E96V, N166S;E96V, F171I; E96V, D203A; E96V, D203G; E96V, T213A; E96V, V225I; E96V,K237E; E96V, V256I; E96V, T258A; E96V, D264G; E96V, T283A; E96V, L289P;E96V, F312L; E96V, T348A; E96V, L380F; E96V, T394M; E96V, F396Y; E96V,R400C; E96V, T413A; E96V, E423D; E96V, L453P; E96V, F458S; E96V, E461G;E96V, F465C; E96V, N467T; E96V, N470S; E96V, A476T; E96V, Q494R; E96V,T496R; E96V, G504S; D101G, G112S; D101G, L114P; D101G, S130L; D101G,K145M; D101G, N166S; D101G, F171I; D101G, D203A; D101G, D203G; D101G,T213A; D101G, V225I; D101G, K237E; D101G, V256I; D101G, T258A; D101G,D264G; D101G, T283A; D101G, L289P; D101G, F312L; D101G, T348A; D101G,L380F; D101G, T394M; D101G, F396Y; D101G, R400C; D101G, T413A; D101G,E423D; D101G, L453P; D101G, F458S; D101G, E461G; D101G, F465C; D101G,N467T; D101G, N470S; D101G, A476T; D101G, Q494R; D101G, T496R; D101G,G504S; G112S, L114P; G112S, S130L; G112S, K145M; G112S, N166S; G112S,F171I; G112S, D203A; G112S, D203G; G112S, T213A; G112S, V225I; G112S,K237E; G112S, V256I; G112S, T258A; G112S, D264G; G112S, T283A; G112S,L289P; G112S, F312L; G112S, T348A; G112S, L380F; G112S, T394M; G112S,F396Y; G112S, R400C; G112S, T413A; G112S, E423D; G112S, L453P; G112S,F458S; G112S, E461G; G112S, F465C; G112S, N467T; G112S, N470S; G112S,A476T; G112S, Q494R; G112S, T496R; G112S, G504S; L114P, S130L; L114P,K145M; L114P, N166S; L114P, F171I; L114P, D203A; L114P, D203G; L114P,T213A; L114P, V225I; L114P, K237E; L114P, V256I; L114P, T258A; L114P,D264G; L114P, T283A; L114P, L289P; L114P, F312L; L114P, T348A; L114P,L380F; L114P, T394M; L114P, F396Y; L114P, R400C; L114P, T413A; L114P,E423D; L114P, L453P; L114P, F458S; L114P, E461G; L114P, F465C; L114P,N467T; L114P, N470S; L114P, A476T; L114P, Q494R; L114P, T496R; L114P,G504S; S130L, K145M; S130L, N166S; S130L, F171I; S130L, D203A; S130L,D203G; S130L, T213A; S130L, V225I; S130L, K237E; S130L, V256I; S130L,T258A; S130L, D264G; S130L, T283A; S130L, L289P; S130L, F312L; S130L,T348A; S130L, L380F; S130L, T394M; S130L, F396Y; S130L, R400C; S130L,T413A; S130L, E423D; S130L, L453P; S130L, F458S; S130L, E461G; S130L,F465C; S130L, N467T; S130L, N470S; S130L, A476T; S130L, Q494R; S130L,T496R; S130L, G504S; K145M, N166S; K145M, F171I; K145M, 203A; K145M,D203G; K145M, T213A; K145M, V225I; K145M, K237E; K145M, V256I; K145M,T258A; K145M, D264G; K145M, T283A; K145M, L289P; K145M, F312L; K145M,T348A; K145M, L380F; K145M, T394M; K145M, F396Y; K145M, R400C; K145M,T413A; K145M, E423D; K145M, L453P; K145M, F458S; K145M, E461G; K145M,F465C; K145M, N467T; K145M, N470S; K145M, A476T; K145M, Q494R; K145M,T496R; K145M, G504S; N166S, F171I; N166S, D203A; N166S, D203G; N166S,T213A; N166S, V225I; N166S, K237E; N166S, V256I; N166S, T258A; N166S,D264G; N166S, T283A; N166S, L289P; N166S, F312L; N166S, T348A; N166S,L380F; N166S, T394M; N166S, F396Y; N166S, R400C; N166S, T413A; N166S,E423D; N166S, L453P; N166S, F458S; N166S, E461G; N166S, F465C; N166S,N467T; N166S, N470S; N166S, A476T; N166S, Q494R; N166S, T496R; N166S,G504S; F171I, D203A; F171I, D203G; F171I, T213A; F171I, V225I; F171I,K237E; F171I, V256I; F171I, T258A; F171I, D264G; F171I, T283A; F171I,L289P; F171I, F312L; F171I, T348A; F171I, L380F; F171I, T394M; F171I,F396Y; F171I, R400C; F171I, T413A; F171I, E423D; F171I, L453P; F171I,F458S; F171I, E461G; F171I, F465C; F171I, N467T; F171I, N470S; F171I,A476T; F171I, Q494R; F171I, T496R; F171I, G504S; D203A, T213A; D203A,V225I; D203A, K237E; D203A, V256I; D203A, T258A; D203A, D264G; D203A,T283A; D203A, L289P; D203A, F312L; D203A, T348A; D203A, L380F; D203A,T394M; D203A, F396Y; D203A, R400C; D203A, T413A; D203A, E423D; D203A,L453P; D203A, F458S; D203A, E461G; D203A, F465C; D203A, N467T; D203A,N470S; D203A, A476T; D203A, Q494R; D203A, T496R; D203A, G504S; D203G,T213A; D203G, V225I; D203G, K237E; D203G, V256I; D203G, T258A; D203G,D264G; D203G, T283A; D203G, L289P; D203G, F312L; D203G, T348A; D203G,L380F; D203G, T394M; D203G, F396Y; D203G, R400C; D203G, T413A; D203G,E423D; D203G, L453P; D203G, F458S; D203G, E461G; D203G, F465C; D203G,N467T; D203G, N470S; D203G, A476T; D203G, Q494R; D203G, T496R; D203G,G504S; T213A, V225I; T213A, K237E; T213A, V256I; T213A, T258A; T213A,D264G; T213A, T283A; T213A, L289P; T213A, F312L; T213A, T348A; T213A,L380F; T213A, T394M; T213A, F396Y; T213A, R400C; T213A, T413A; T213A,E423D; T213A, L453P; T213A, F458S; T213A, E461G; T213A, F465C; T213A,N467T; T213A, N470S; T213A, A476T; T213A, Q494R; T213A, T496R; T213A,G504S; V225I, K237E; V225I, V256I; V225I, T258A; V225I, D264G; V225I,T283A; V225I, L289P; V225I, F312L; V225I, T348A; V225I, L380F; V225I,T394M; V225I, F396Y; V225I, R400C; V225I, T413A; V225I, E423D; V225I,L453P; V225I, F458S; V225I, E461G; V225I, F465C; V225I, N467T; V225I,N470S; V225I, A476T; V225I, Q494R; V225I, T496R; V225I, G504S; K237E,V256I; K237E, T258A; K237E, D264G; K237E, T283A; K237E, L289P; K237E,F312L; K237E, T348A; K237E, L380F; K237E, T394M; K237E, F396Y; K237E,R400C; K237E, T413A; K237E, E423D; K237E, L453P; K237E, F458S; K237E,E461G; K237E, F465C; K237E, N467T; K237E, N470S; K237E, A476T; K237E,Q494R; K237E, T496R; K237E, G504S; V256I, T258A; V256I, D264G; V256I,T283A; V256I, L289P; V256I, F312L; V256I, T348A; V256I, L380F; V256I,T394M; V256I, F396Y; V256I, R400C; V256I, T413A; V256I, E423D; V256I,L453P; V256I, F458S; V256I, E461G; V256I, F465C; V256I, N467T; V256I,N470S; V256I, A476T; V256I, Q494R; V256I, T496R; V256I, G504S; T258A,D264G; T258A, T283A; T258A, L289P; T258A, F312L; T258A, T348A; T258A,L380F; T258A, T394M; T258A, F396Y; T258A, R400C; T258A, T413A; T258A,E423D; T258A, L453P; T258A, F458S; T258A, E461G; T258A, F465C; T258A,N467T; T258A, N470S; T258A, A476T; T258A, Q494R; T258A, T496R; T258A,G504S; D264G, T283A; D264G, L289P; D264G, F312L; D264G, T348A; D264G,L380F; D264G, T394M; D264G, F396Y; D264G, R400C; D264G, T413A; D264G,E423D; D264G, L453P; D264G, F458S; D264G, E461G; D264G, F465C; D264G,N467T; D264G, N470S; D264G, A476T; D264G, Q494R; D264G, T496R; D264G,G504S; T283A, L289P; T283A, F312L; T283A, T348A; T283A, L380F; T283A,T394M; T283A, F396Y; T283A, R400C; T283A, T413A; T283A, E423D; T283A,L453P; T283A, F458S; T283A, E461G; T283A, F465C; T283A, N467T; T283A,N470S; T283A, A476T; T283A, Q494R; T283A, T496R; T283A, G504S; L289P,F312L; L289P, T348A; L289P, L380F; L289P, T394M; L289P, F396Y; L289P,R400C; L289P, T413A; L289P, E423D; L289P, L453P; L289P, F458S; L289P,E461G; L289P, F465C; L289P, N467T; L289P, N470S; L289P, A476T; L289P,Q494R; L289P, T496R; L289P, G504S; F312L, T348A; F312L, L380F; F312L,T394M; F312L, F396Y; F312L, R400C; F312L, T413A; F312L, E423D; F312L,L453P; F312L, F458S; F312L, E461G; F312L, F465C; F312L, N467T; F312L,N470S; F312L, A476T; F312L, Q494R; F312L, T496R; F312L, G504S; T348A,L380F; T348A, T394M; T348A, F396Y; T348A, R400C; T348A, T413A; T348A,E423D; T348A, L453P; T348A, 458S; T348A, E461G; T348A, F465C; T348A,N467T; T348A, N470S; T348A, A476T; T348A, Q494R; T348A, T496R; T348A,G504S; L380F, T394M; L380F, F396Y; L380F, R400C; L380F, T413A; L380F,E423D; L380F, L453P; L380F, F458S; L380F, E461G; L380F, F465C; L380F,N467T; L380F, N470S; L380F, A476T; L380F, Q494R; L380F, T496R; L380F,G504S; T394M, F396Y; T394M, R400C; T394M, T413A; T394M, E423D; T394M,L453P; T394M, F458S; T394M, E461G; T394M, F465C; T394M, N467T; T394M,N470S; T394M, A476T; T394M, Q494R; T394M, T496R; T394M, G504S; F396Y,R400C; F396Y, T413A; F396Y, E423D; F396Y, L453P; F396Y, F458S; F396Y,E461G; F396Y, F465C; F396Y, N467T; F396Y, N470S; F396Y, A476T; F396Y,Q494R; F396Y, T496R; F396Y, G504S; R400C, T413A; R400C, E423D; R400C,L453P; R400C, F458S; R400C, E461G; R400C, F465C; R400C, N467T; R400C,N470S; R400C, A476T; R400C, Q494R; R400C, T496R; R400C, 504S; T413A,E423D; T413A, L453P; T413A, F458S; T413A, E461G; T413A, F465C; T413A,N467T; T413A, N470S; T413A, A476T; T413A, Q494R; T413A, T496R; T413A,G504S; E423D, L453P; E423D, F458S; E423D, E461G; E423D, F465C; E423D,N467T; E423D, N470S; E423D, A476T; E423D, Q494R; E423D, T496R; E423D,G504S; L453P, F458S; L453P, E461G; L453P, F465C; L453P, N467T; L453P,N470S; L453P, A476T; L453P, Q494R; L453P, T496R; L453P, G504S; F458S,E461G; F458S, F465C; F458S, N467T; F458S, N470S; F458S, A476T; F458S,Q494R; F458S, T496R; F458S, G504S; E461G, F465C; E461G, N467T; E461G,N470S; E461G, A476T; E461G, Q494R; E461G, T496R; E461G, G504S; F465C,N467T; F465C, N470S; F465C, A476T; F465C, Q494R; F465C, T496R; F465C,G504S; N467T, N470S; N467T, A476T; N467T, Q494R; N467T, T496R; N467T,G504S; N470S, A476T; N470S, Q494R; N470S, T496R; N470S, G504S; A476T,Q494R; A476T, T496R; A476T, G504S; Q494R, T496R; Q494R, G504S; T496R,G504S; S2P, M71V, E96V; S2P, M71V, D101G; S2P, M71V, G112S; S2P, M71V,L114P; S2P, M71V, S130L; S2P, M71V, K145M; S2P, M71V, N166S; S2P, M71V,F171I; S2P, M71V, D203A; S2P, M71V, D203G; S2P, M71V, T213A; S2P, M71V,V225I; S2P, M71V, K237E; S2P, M71V, V256I; S2P, M71V, T258A; S2P, M71V,D264G; S2P, M71V, T283A; S2P, M71V, L289P; S2P, M71V, F312L; S2P, M71V,T348A; S2P, M71V, L380F; S2P, M71V, T394M; S2P, M71V, F396Y; S2P, M71V,R400C; S2P, M71V, T413A; S2P, M71V, E423D; S2P, M71V, L453P; S2P, M71V,F458S; S2P, M71V, E461G; S2P, M71V, F465C; S2P, M71V, N467T; S2P, M71V,N470S; S2P, M71V, A476T; S2P, M71V, Q494R; S2P, M71V, T496R; S2P, M71V,G504S; S2P, M71T, E96V; S2P, M71T, D101G; S2P, M71T, G112S; S2P, M71T,L114P; S2P, M71T, S130L; S2P, M71T, K145M; S2P, M71T, N166S; S2P, M71T,F171I; S2P, M71T, D203A; S2P, M71T, D203G; S2P, M71T, T213A; S2P, M71T,V225I; S2P, M71T, K237E; S2P, M71T, V256I; S2P, M71T, T258A; S2P, M71T,D264G; S2P, M71T, T283A; S2P, M71T, L289P; S2P, M71T, F312L; S2P, M71T,T348A; S2P, M71T, L380F; S2P, M71T, T394M; S2P, M71T, 396Y; S2P, M71T,R400C; S2P, M71T, T413A; S2P, M71T, E423D; S2P, M71T, L453P; S2P, M71T,F458S; S2P, M71T, E461G; S2P, M71T, F465C; S2P, M71T, N467T; S2P, M71T,N470S; S2P, M71T, A476T; S2P, M71T, Q494R; S2P, M71T, T496R; S2P, M71T,G504S; S2P, M71A, E96V; S2P, M71A, D101G; S2P, M71A, G112S; S2P, M71A,L114P; S2P, M71A, S130L; S2P, M71A, K145M; S2P, M71A, N166S; S2P, M71A,F171I; S2P, M71A, D203A; S2P, M71A, D203G; S2P, M71A, T213A; S2P, M71A,V225I; S2P, M71A, K237E; S2P, M71A, V256I; S2P, M71A, T258A; S2P, M71A,D264G; S2P, M71A, T283A; S2P, M71A, L289P; S2P, M71A, F312L; S2P, M71A,T348A; S2P, M71A, L380F; S2P, M71A, T394M; S2P, M71A, F396Y; S2P, M71A,R400C; S2P, M71A, T413A; S2P, M71A, E423D; S2P, M71A, L453P; S2P, M71A,F458S; S2P, M71A, E461G; S2P, M71A, F465C; S2P, M71A, N467T; S2P, M71A,N470S; S2P, M71A, A476T; S2P, M71A, Q494R; S2P, M71A, T496R; S2P, M71A,G504S; S2P, M71C, E96V; S2P, M71C, D101G; S2P, M71C, G112S; S2P, M71C,L114P; S2P, M71C, S130L; S2P, M71C, K145M; S2P, M71C, N166S; S2P, M71C,F171I; S2P, M71C, D203A; S2P, M71C, D203G; S2P, M71C, T213A; S2P, M71C,V225I; S2P, M71C, K237E; S2P, M71C, V256I; S2P, M71C, T258A; S2P, M71C,D264G; S2P, M71C, T283A; S2P, M71C, L289P; S2P, M71C, F312L; S2P, M71C,T348A; S2P, M71C, L380F; S2P, M71C, T394M; S2P, M71C, F396Y; S2P, M71C,R400C; S2P, M71C, T413A; S2P, M71C, E423D; S2P, M71C, L453P; S2P, M71C,F458S; S2P, M71C, E461G; S2P, M71C, F465C; S2P, M71C, N467T; S2P, M71C,N470S; S2P, M71C, A476T; S2P, M71C, Q494R; S2P, M71C, T496R; S2P, M71C,G504S; S2P, M71V, N75K; S2P, M71T, N75K; S2P, M71A, N75K; S2P, M71C,N75K; S2P, N75K, E96V; S2P, N75K, D101G; S2P, N75K, G112S; S2P, N75K,L114P; S2P, N75K, S130L; S2P, N75K, K145M; S2P, N75K, N166S; S2P, N75K,F171I; S2P, N75K, 203A; S2P, N75K, D203G; S2P, N75K, T213A; S2P, N75K,V225I; S2P, N75K, K237E; S2P, N75K, V256I; S2P, N75K, T258A; S2P, N75K,D264G; S2P, N75K, T283A; S2P, N75K, L289P; S2P, N75K, F312L; S2P, N75K,T348A; S2P, N75K, L380F; S2P, N75K, T394M; S2P, N75K, 396Y; S2P, N75K,R400C; S2P, N75K, T413A; S2P, N75K, E423D; S2P, N75K, L453P; S2P, N75K,F458S; S2P, N75K, E461G; S2P, N75K, F465C; S2P, N75K, N467T; S2P, N75K,470S; S2P, N75K, A476T; S2P, N75K, Q494R; S2P, N75K, T496R; S2P, N75K,G504S; M71V, 75K, E96V; M71V, N75K, D101G; M71V, N75K, G112S; M71V,N75K, L114P; M71V, N75K, S130L; M71V, N75K, K145M; M71V, N75K, N166S;M71V, N75K, F171I; M71V, N75K, D203A; M71V, N75K, D203G; M71V, N75K,T213A; M71V, N75K, V225I; M71V, N75K, K237E; M71V, N75K, V256I; M71V,N75K, T258A; M71V, N75K, D264G; M71V, N75K, T283A; M71V, N75K, L289P;M71V, N75K, F312L; M71V, N75K, T348A; M71V, N75K, L380F; M71V, N75K,T394M; M71V, N75K, F396Y; M71V, N75K, R400C; M71V, N75K, T413A; M71V,N75K, E423D; M71V, N75K, L453P; M71V, N75K, F458S; M71V, N75K, E461G;M71V, N75K, F465C; M71V, N75K, N467T; M71V, N75K, N470S; M71V, N75K,A476T; M71V, N75K, Q494R; M71V, N75K, T496R; M71V, N75K, G504S; M71T,N75K, E96V; M71T, N75K, D101G; M71T, N75K, G112S; M71T, N75K, L114P;M71T, N75K, S130L; M71T, N75K, K145M; M71T, N75K, N166S; M71T, N75K,F171I; M71T, N75K, D203A; M71T, N75K, D203G; M71T, N75K, T213A; M71T,N75K, V225I; M71T, N75K, K237E; M71T, N75K, V256I; M71T, N75K, T258A;M71T, N75K, D264G; M71T, N75K, T283A; M71T, N75K, L289P; M71T, N75K,F312L; M71T, N75K, T348A; M71T, N75K, L380F; M71T, N75K, T394M; M71T,N75K, F396Y; M71T, N75K, R400C; M71T, N75K, T413A; M71T, N75K, E423D;M71T, N75K, L453P; M71T, N75K, F458S; M71T, N75K, E461G; M71T, N75K,F465C; M71T, N75K, N467T; M71T, N75K, N470S; M71T, N75K, A476T; M71T,N75K, Q494R; M71T, N75K, T496R; M71T, N75K, G504S; M71A, N75K, E96V;M71A, N75K, D101G; M71A, N75K, G112S; M71A, N75K, L114P; M71A, N75K,S130L; M71A, N75K, K145M; M71A, N75K, N166S; M71A, N75K, F171I; M71A,N75K, D203A; M71A, N75K, D203G; M71A, N75K, T213A; M71A, N75K, V225I;M71A, N75K, K237E; M71A, N75K, V256I; M71A, N75K, T258A; M71A, N75K,D264G; M71A, N75K, T283A; M71A, N75K, L289P; M71A, N75K, F312L; M71A,N75K, T348A; M71A, N75K, L380F; M71A, N75K, T394M; M71A, N75K, F396Y;M71A, N75K, R400C; M71A, N75K, T413A; M71A, N75K, E423D; M71A, N75K,L453P; M71A, N75K, F458S; M71A, N75K, E461G; M71A, N75K, F465C; M71A,N75K, N467T; M71A, N75K, N470S; M71A, N75K, A476T; M71A, N75K, Q494R;M71A, N75K, T496R; M71A, N75K, G504S; M71C, N75K, E96V; M71C, N75K,D101G; M71C, N75K, G112S; M71C, N75K, L114P; M71C, N75K, S130L; M71C,N75K, K145M; M71C, N75K, N166S; M71C, N75K, F171I; M71C, N75K, D203A;M71C, N75K, D203G; M71C, N75K, T213A; M71C, N75K, V225I; M71C, N75K,K237E; M71C, N75K, V256I; M71C, N75K, T258A; M71C, N75K, D264G; M71C,N75K, T283A; M71C, N75K, L289P; M71C, N75K, F312L; M71C, N75K, T348A;M71C, N75K, L380F; M71C, N75K, T394M; M71C, N75K, F396Y; M71C, N75K,R400C; M71C, N75K, T413A; M71C, N75K, E423D; M71C, N75K, L453P; M71C,N75K, F458S; M71C, N75K, E461G; M71C, N75K, F465C; M71C, N75K, N467T;M71C, N75K, N470S; M71C, N75K, A476T; M71C, N75K, Q494R; M71C, N75K,T496R; M71C, N75K, G504S; N75K, E96V, D101G; N75K, E96V, G112S; N75K,E96V, L114P; N75K, E96V, S130L; N75K, E96V, K145M; N75K, E96V, N166S;N75K, E96V, F171I; N75K, E96V, D203A; N75K, E96V, D203G; N75K, E96V,T213A; N75K, E96V, V225I; N75K, E96V, K237E; N75K, E96V, V256I; N75K,E96V, T258A; N75K, E96V, D264G; N75K, E96V, T283A; N75K, E96V, L289P;N75K, E96V, F312L; N75K, E96V, T348A; N75K, E96V, L380F; N75K, E96V,T394M; N75K, E96V, F396Y; N75K, E96V, R400C; N75K, E96V, T413A; N75K,E96V, E423D; N75K, E96V, L453P; N75K, E96V, F458S; N75K, E96V, E461G;N75K, E96V, F465C; N75K, E96V, N467T; N75K, E96V, N470S; N75K, E96V,A476T; N75K, E96V, Q494R; N75K, E96V, T496R; N75K, E96V, G504S; E96V,D101G, G112S; E96V, D101G, L114P; E96V, D101G, S130L; E96V, D101G,K145M; E96V, D101G, N166S; E96V, D101G, F171I; E96V, D101G, D203A; E96V,D101G, D203G; E96V, D101G, T213A; E96V, D101G, V225I; E96V, D101G,K237E; E96V, D101G, V256I; E96V, D101G, T258A; E96V, D101G, D264G; E96V,D101G, T283A; E96V, D101G, L289P; E96V, D101G, F312L; E96V, D101G,T348A; E96V, D101G, L380F; E96V, D101G, T394M; E96V, D101G, F396Y; E96V,D101G, R400C; E96V, D101G, T413A; E96V, D101G, E423D; E96V, D101G,L453P; E96V, D101G, F458S; E96V, D101G, E461G; E96V, D101G, F465C; E96V,D101G, N467T; E96V, D101G, N470S; E96V, D101G, A476T; E96V, D101G,Q494R; E96V, D101G, T496R; E96V, D101G, G504S; D101G, G112S, L114P;D101G, G112S, S130L; D101G, G112S, K145M; D101G, G112S, N166S; D101G,G112S, F171I; D101G, G112S, D203A; D101G, G112S, D203G; D101G, G112S,T213A; D101G, G112S, V225I; D101G, G112S, K237E; D101G, G112S, V256I;D101G, G112S, T258A; D101G, G112S, D264G; D101G, G112S, T283A; D101G,G112S, L289P; D101G, G112S, F312L; D101G, G112S, T348A; D101G, G112S,L380F; D101G, G112S, T394M; D101G, G112S, F396Y; D101G, G112S, R400C;D101G, G112S, T413A; D101G, G112S, E423D; D101G, G112S, L453P; D101G,G112S, F458S; D101G, G112S, E461G; D101G, G112S, F465C; D101G, G112S,N467T; D101G, G112S, N470S; D101G, G112S, A476T; D101G, G112S, Q494R;D101G, G112S, T496R; D101G, G112S, G504S; G112S, L114P, S130L; G112S,L114P, K145M; G112S, L114P, N166S; G112S, L114P, F171I; G112S, L114P,D203A; G112S, L114P, D203G; G112S, L114P, T213A; G112S, L114P, V225I;G112S, L114P, K237E; G112S, L114P, V256I; G112S, L114P, T258A; G112S,L114P, D264G; G112S, L114P, T283A; G112S, L114P, L289P; G112S, L114P,F312L; G112S, L114P, T348A; G112S, L114P, L380F; G112S, L114P, T394M;G112S, L114P, F396Y; G112S, L114P, R400C; G112S, L114P, T413A; G112S,L114P, E423D; G112S, L114P, L453P; G112S, L114P, F458S; G112S, L114P,E461G; G112S, L114P, F465C; G112S, L114P, N467T; G112S, L114P, N470S;G112S, L114P, A476T; G112S, L114P, Q494R; G112S, L114P, T496R; G112S,L114P, G504S; L114P, S130L, K145M; L114P, S130L, N166S; L114P, S130L,F171I, L114P, S130L, D203A; L114P, S130L, D203G; L114P, S130L, T213A;L114P, S130L, V225I; L114P, S130L, K237E; L114P, S130L, V256I; L114P,S130L, T258A; L114P, S130L, D264G; L114P, S130L, T283A; L114P, S130L,L289P; L114P, S130L, F312L; L114P, S130L, T348A; L114P, S130L, L380F;L114P, S130L, T394M; L114P, S130L, F396Y; L114P, S130L, R400C; L114P,S130L, T413A; L114P, S130L, E423D; L114P, S130L, L453P; L114P, S130L,F458S; L114P, S130L, E461G; L114P, S130L, F465C; L114P, S130L, N467T;L114P, S130L, N470S; L114P, S130L, A476T; L114P, S130L, Q494R; L114P,S130L, T496R; L114P, S130L, G504S; S130L, K145M, N166S; S130L, K145M,F171I; S130L, K145M, D203A; S130L, K145M, D203G; S130L, K145M, T213A;S130L, K145M, V225I; S130L, K145M, K237E; S130L, K145M, V256I; S130L,K145M, T258A; S130L, K145M, D264G; S130L, K145M, T283A; S130L, K145M,L289P; S130L, K145M, F312L; S130L, K145M, T348A; S130L, K145M, L380F;S130L, K145M, T394M; S130L, K145M, F396Y; S130L, K145M, R400C; S130L,K145M, T413A; S130L, K145M, E423D; S130L, K145M, L453P; S130L, K145M,F458S; S130L, K145M, E461G; S130L, K145M, F465C; S130L, K145M, N467T;S130L, K145M, N470S; S130L K145M, A476T; S130L, K145M, Q494R; S130L,K145M, T496R; S130L, K145M, G504S; K145M, N166S, F171I; K145M, N166S,D203A; K145M, N166S, D203G; K145M, N166S, T213A; K145M, N166S, V225I;K145M, N166S, K237E; K145M, N166S, V256I; K145M, N166S, T258A; K145M,N166S, D264G; K145M, N166S, T283A; K145M, N166S, L289P; K145M, N166S,F312L; K145M, N166S, T348A; K145M, N166S, L380F; K145M, N166S, 394M;K145M, N166S, F396Y; K145M, N166S, R400C; K145M, N166S, T413A; K145M,N166S, E423D; K145M, N166S, L453P; K145M, N166S, F458S; K145M, N166S,E461G; K145M, N166S, F465C; K145M, N166S, N467T; K145M, N166S, N470S;K145M, N166S, A476T; K145M, N166S, Q494R; K145M, N166S, T496R; K145M,N166S, G504S; N166S, F171I, D203A; N166S, F171I, D203G; N166S, F171I,T213A; N166S, F171I, V225I; N166S, F171I, K237E; N166S, F171I, V256I;N166S, F171I, T258A; N166S, F171I, D264G; N166S, F171I, T283A; N166S,F171I, L289P; N166S, F171I, F312L; N166S, F171I, T348A; N166S, F171I,L380F; N166S, F171I, T394M; N166S, F171I, F396Y; N166S, F171I, R400C;N166S, F171I, T413A; N166S, F171I, E423D; N166S, F171I, L453P; N166S,F171I, F458S; N166S, F171I, E461G; N166S, F171I, F465C; N166S, F171I,N467T; N166S, F171I, N470S; N166S, F171I, A476T; N166S, F171I, Q494R;N166S, F171I, T496R; N166S, F171I, G504S; F171I, D203A, D203G; F171I,D203A, T213A; F171I, D203A, V225I; F171I, D203A, K237E; F171I, D203A,V256I; F171I, D203A, T258A; F171I, D203A, D264G; F171I, D203A, T283A;F171I, D203A, L289P; F171I, D203A, F312L; F171I, D203A, T348A; F171I,D203A, L380F; F171I, D203A, T394M; F171I, D203A, F396Y; F171I, D203A,R400C; F171I, D203A, T413A; F171I, D203A, E423D; F171I, D203A, L453P;F171I, D203A, F458S; F171I, D203A, E461G; F171I, D203A, F465C; F171I,D203A, N467T; F171I, D203A, N470S; F171I, D203A, A476T; F171I, D203A,Q494R; F171I, D203A, T496R; F171I, D203A, G504S; D203A, T213A, V225I;D203A, T213A, K237E; D203A, T213A, V256I; D203A, T213A, T258A; D203A,T213A, D264G; D203A, T213A, T283A; D203A, T213A, L289P; D203A, T213A,F312L; D203A, T213A, T348A; D203A, T213A, L380F; D203A, T213A, T394M;D203A, T213A, F396Y; D203A, T213A, R400C; D203A, T213A, T413A; D203A,T213A, E423D; D203A, T213A, L453P; D203A, T213A, F458S; D203A, T213A,E461G; D203A, T213A, F465C; D203A, T213A, N467T; D203A, T213A, N470S;D203A, T213A, A476T; D203A, T213A, Q494R; D203A, T213A, T496R; D203A,T213A, G504S; D203C, T213A, V225I; D203C, T213A, K237E; D203C, T213A,V256I; D203C, T213A, T258A; D203C, T213A, D264G; D203C, T213A, T283A;D203C, T213A, L289P; D203C, T213A, F312L; D203C, T213A, T348A; D203C,T213A, L380F; D203C, T213A, T394M; D203C, T213A, F396Y; D203C, T213A,R400C; D203C, T213A, T413A; D203C, T213A, E423D; D203C, T213A, L453P;D203C, T213A, F458S; D203C, T213A, E461G; D203C, T213A, F465C; D203C,T213A, N467T; D203C, T213A, N470S; D203C, T213A, A476T; D203C, T213A,Q494R; D203C, T213A, T496R; D203C, T213A, G504S; T213A, V225I, K237E;T213A, V225I, V256I; T213A, V225I, T258A; T213A, V225I, D264G; T213A,V225I, T283A; T213A, V225I, L289P; T213A, V225I, F312L; T213A, V225I,T348A; T213A, V225I, L380F; T213A, V225I, T394M; T213A, V225I, F396Y;T213A, V225I, R400C; T213A, V225I, T413A; T213A, V225I, E423D; T213A,V225I, L453P; T213A, V225I, F458S; T213A, V225I, E461G; T213A, V225I,F465C; T213A, V225I, N467T; T213A, V225I, N470S; T213A, V225I, A476T;T213A, V225I, Q494R; T213A, V225I, T496R; T213A, V225I, G504S; V225I,K237E, V256I; V225I, K237E, T258A; V225I, K237E, D264G; V225I, K237E,T283A; V225I, K237E, L289P; V225I, K237E, F312L; V225I, K237E, T348A;V225I, K237E, L380F; V225I, K237E, T394M; V225I, K237E, F396Y; V225I,K237E, R400C; V225I, K237E, T413A; V225I, K237E, E423D; V225I, K237E,L453P; V225I, K237E, F458S; V225I, K237E, E461G; V225I, K237E, F465C;V225I, K237E, N467T; V225I, K237E, N470S; V225I, K237E, A476T; V225I,K237E, Q494R; V225I, K237E, T496R; V225I, K237E, G504S; K237E, V256I,T258A; K237E, V256I, D264G; K237E, V256I, T283A; K237E, V256I, L289P;K237E, V256I, F312L; K237E, V256I, T348A; K237E, V256I, L380F; K237E,V256I, T394M; K237E, V256I, F396Y; K237E, V256I, R400C; K237E, V256I,T413A; K237E, V256I, E423D; K237E, V256I, L453P; K237E, V256I, F458S;K237E, V256I, E461G; K237E, V256I, F465C; K237E, V256I, N467T; K237E,V256I, N470S; K237E, V256I, A476T; K237E, V256I, Q494R; K237E, V256I,T496R; K237E, V256I, G504S; V256I, T258A, D264G; V256I, T258A, T283A;V256I, T258A, L289P; V256I, T258A, F312L; V256I, T258A, T348A; V256I,T258A, L380F; V256I, T258A, T394M; V256I, T258A, F396Y; V256I, T258A,R400C; V256I, T258A, T413A; V256I, T258A, E423D; V256I, T258A, L453P;V256I, T258A, F458S; V256I, T258A, E461G; V256I, T258A, F465C; V256I,T258A, N467T; V256I, T258A, N470S; V256I, T258A, A476T; V256I, T258A,Q494R; V256I, T258A, T496R; V256I, T258A, G504S; T258A, D264G, T283A;T258A, D264G, L289P; T258A, D264G, F312L; T258A, D264G, T348A; T258A,D264G, L380F; T258A, D264G, T394M; T258A, D264G, F396Y; T258A, D264G,R400C; T258A, D264G, T413A; T258A, D264G, E423D; T258A, D264G, L453P;T258A, D264G, F458S; T258A, D264G, E461G; T258A, D264G, F465C; T258A,D264G, N467T; T258A, D264G, N470S; T258A, D264G, A476T; T258A, D264G,Q494R; T258A, D264G, T496R; T258A, D264G, G504S; D264G, T283A, L289P;D264G, T283A, F312L; D264G, T283A, T348A; D264G, T283A, L380F; D264G,T283A, T394M; D264G, T283A, F396Y; D264G, T283A, R400C; D264G, T283A,T413A; D264G, T283A, E423D; D264G, T283A, L453P; D264G, T283A, F458S;D264G, T283A, E461G; D264G, T283A, F465C; D264G, T283A, N467T; D264G,T283A, N470S; D264G, T283A, A476T; D264G, T283A, Q494R; D264G, T283A,T496R; D264G, T283A, G504S; T283A, L289P, F312L; T283A, L289P, T348A;T283A, L289P, L380F; T283A, L289P, T394M; T283A, L289P, F396Y; T283A,L289P, R400C; T283A, L289P, T413A; T283A, L289P, E423D; T283A, L289P,L453P; T283A, L289P, F458S; T283A, L289P, E461G; T283A, L289P, F465C;T283A, L289P, N467T; T283A, L289P, N470S; T283A, L289P, A476T; T283A,L289P, Q494R; T283A, L289P, T496R; T283A, L289P, G504S; L289P, F312L,T348A; L289P, F312L, L380F; L289P, F312L, T394M; L289P, F312L, F396Y;L289P, F312L, R400C; L289P, F312L, T413A; L289P, F312L, E423D; L289P,F312L, L453P; L289P, F312L, F458S; L289P, F312L, E461G; L289P, F312L,F465C; L289P, F312L, N467T; L289P, F312L, N470S; L289P, F312L, A476T;L289P, F312L, Q494R; L289P, F312L, T496R; L289P, F312L, G504S; F312L,T348A, L380F; F312L, T348A, T394M; F312L, T348A, F396Y; F312L, T348A,R400C; F312L, T348A, T413A; F312L, T348A, E423D; F312L, T348A, L453P;F312L, T348A, F458S; F312L, T348A, E461G; F312L, T348A, F465C; F312L,T348A, N467T; F312L, T348A, N470S; F312L, T348A, A476T; F312L, T348A,Q494R; F312L, T348A, T496R; F312L, T348A, G504S; T348A, L380F, T394M;T348A, L380F, F396Y; T348A, L380F, R400C; T348A, L380F, T413A; T348A,L380F, E423D; T348A, L380F, L453P; T348A, L380F, F458S; T348A, L380F,E461G; T348A, L380F, F465C; T348A, L380F, N467T; T348A, L380F, N470S;T348A, L380F, A476T; T348A, L380F, Q494R; T348A, L380F, T496R; T348A,L380F, G504S; L380F, T394M, F396Y; L380F, T394M, R400C; L380F, T394M,T413A; L380F, T394M, E423D; L380F, T394M, L453P; L380F, T394M, F458S;L380F, T394M, E461G; L380F, T394M, F465C; L380F, T394M, N467T; L380F,T394M, N470S; L380F, T394M, A476T; L380F, T394M, Q494R; L380F, T394M,T496R; L380F, T394M, G504S; T394M, F396Y, R400C; T394M, F396Y, T413A;T394M, F396Y, E423D; T394M, F396Y, L453P; T394M, F396Y, F458S; T394M,F396Y, E461G; T394M, F396Y, F465C; T394M, F396Y, N467T; T394M, F396Y,N470S; T394M, F396Y, A476T; T394M, F396Y, Q494R; T394M, F396Y, T496R;T394M, F396Y, G504S; F396Y, R400C, T413A; F396Y, R400C, E423D; F396Y,R400C, L453P; F396Y, R400C, F458S; F396Y, R400C, E461G; F396Y, R400C,F465C; F396Y, R400C, N467T; F396Y, R400C, N470S; F396Y, R400C, A476T;F396Y, R400C, Q494R; F396Y, R400C, T496R; F396Y, R400C, G504S; R400C,T413A, E423D; R400C, T413A, L453P; R400C, T413A, F458S; R400C, T413A,E461G; R400C, T413A, F465C; R400C, T413A, N467T; R400C, T413A, N470S;R400C, T413A, A476T; R400C, T413A, Q494R; R400C, T413A, T496R; R400C,T413A, G504S; T413A, E423D, L453P; T413A, E423D, F458S; T413A, E423D,E461G; T413A, E423D, F465C; T413A, E423D, N467T; T413A, E423D, N470S;T413A, E423D, A476T; T413A, E423D, Q494R; T413A, E423D, T496R; T413A,E423D, G504S; E423D, L453P, F458S; E423D, L453P, E461G; E423D, L453P,F465C; E423D, L453P, N467T; E423D, L453P, N470S; E423D, L453P, A476T;E423D, L453P, Q494R; E423D, L453P, T496R; E423D, L453P, G504S; L453P,F458S, E461G; L453P, F458S, F465C; L453P, F458S, N467T; L453P, F458S,N470S; L453P, F458S, A476T; L453P, F458S, Q494R; L453P, F458S, T496R;L453P, F458S, G504S; F458S, E461G, F465C; F458S, E461G, N467T; F458S,E461G, N470S; F458S, E461G, A476T; F458S, E461G, Q494R; F458S, E461G,T496R; F458S, E461G, G504S; E461G, F465C, N467T; E461G, F465C, N470S;E461G, F465C, A476T; E461G, F465C, Q494R; E461G, F465C, T496R; E461G,F465C, G504S; F465C, N467T, N470S; F465C, N467T, A476T; F465C, N467T,Q494R; F465C, N467T, T496R; F465C, N467T, G504S; N467T, N470S, A476T;N467T, N470S, Q494R; N467T, N470S, T496R; N467T, N470S, G504S; N470S,A476T, Q494R; N470S, A476T, T496R; N470S, A476T, G504S; A476T, Q494R,T496R; A476T, Q494R, G504S; Q494R, T496R, G504S; S2P, M71V, N75K, E96V;S2P, M71V, E96V, D101G; S2P, M71V, E96V, G112S; S2P, M71V, E96V, L114P;S2P, M71V, E96V, S130L; S2P, M71V, E96V, K145M; S2P, M71V, E96V, N166S;S2P, M71V, E96V, F171I; S2P, M71V, E96V, D203A; S2P, M71V, E96V, D203G;S2P, M71V, E96V, T213A; S2P, M71V, E96V, V225I; S2P, M71V, E96V, K237E;S2P, M71V, E96V, V256I; S2P, M71V, E96V, T258A; S2P, M71V, E96V, D264G;S2P, M71V, E96V, T283A; S2P, M71V, E96V, L289P; S2P, M71V, E96V, F312L;S2P, M71V, E96V, T348A; S2P, M71V, E96V, L380F; S2P, M71V, E96V, T394M;S2P, M71V, E96V, F396Y; S2P, M71V, E96V, R400C; S2P, M71V, E96V, T413A;S2P, M71V, E96V, E423D; S2P, M71V, E96V, L453P; S2P, M71V, E96V, F458S;S2P, M71V, E96V, E461G; S2P, M71V, E96V, F465C; S2P, M71V, E96V, N467T;S2P, M71V, E96V, N470S; S2P, M71V, E96V, A476T; S2P, M71V, E96V, Q494R;S2P, M71V, E96V, T496R; S2P, M71V, E96V, G504S; S2P, M71T, E96V, D101G;S2P, M71T, E96V, G112S; S2P, M71T, E96V, L114P; S2P, M71T, E96V, S130L;S2P, M71T, E96V, K145M; S2P, M71T, E96V, N166S; S2P, M71T, E96V, F171I;S2P, M71T, E96V, D203A; S2P, M71T, E96V, D203G; S2P, M71T, E96V, T213A;S2P, M71T, E96V, V225I; S2P, M71T, E96V, K237E; S2P, M71T, E96V, V256I;S2P, M71T, E96V, T258A; S2P, M71T, E96V, D264G; S2P, M71T, E96V, T283A;S2P, M71T, E96V, L289P; S2P, M71T, E96V, F312L; S2P, M71T, E96V, T348A;S2P, M71T, E96V, L380F; S2P, M71T, E96V, T394M; S2P, M71T, E96V, F396Y;S2P, M71T, E96V, R400C; S2P, M71T, E96V, T413A; S2P, M71T, E96V, E423D;S2P, M71T, E96V, L453P; S2P, M71T, E96V, F458S; S2P, M71T, E96V, E461G;S2P, M71T, E96V, F465C; S2P, M71T, E96V, N467T; S2P, M71T, E96V, N470S;S2P, M71T, E96V, A476T; S2P, M71T, E96V, Q494R; S2P, M71T, E96V, T496R;S2P, M71T, E96V, G504S; S2P, M71A, E96V, D101G; S2P, M71A, E96V, G112S;S2P, M71A, E96V, L114P; S2P, M71A, E96V, S130L; S2P, M71A, E96V, K145M;S2P, M71A, E96V, N166S; S2P, M71A, E96V, F171I; S2P, M71A, E96V, D203A;S2P, M71A, E96V, D203G; S2P, M71A, E96V, T213A; S2P, M71A, E96V, V225I;S2P, M71A, E96V, K237E; S2P, M71A, E96V, V256I; S2P, M71A, E96V, T258A;S2P, M71A, E96V, D264G; S2P, M71A, E96V, T283A; S2P, M71A, E96V, L289P;S2P, M71A, E96V, F312L; S2P, M71A, E96V, T348A; S2P, M71A, E96V, L380F;S2P, M71A, E96V, T394M; S2P, M71A, E96V, F396Y; S2P, M71A, E96V, R400C;S2P, M71A, E96V, T413A; S2P, M71A, E96V, E423D; S2P, M71A, E96V, L453P;S2P, M71A, E96V, F458S; S2P, M71A, E96V, E461G; S2P, M71A, E96V, F465C;S2P, M71A, E96V, N467T; S2P, M71A, E96V, N470S; S2P, M71A, E96V, A476T;S2P, M71A, E96V, Q494R; S2P, M71A, E96V, T496R; S2P, M71A, E96V, G504S;S2P, M71C, E96V, D101G; S2P, M71C, E96V, G112S; S2P, M71C, E96V, L114P;S2P, M71C, E96V, S130L; S2P, M71C, E96V, K145M; S2P, M71C, E96V, N166S;S2P, M71C, E96V, F171I; S2P, M71C, E96V, D203A; S2P, M71C, E96V, D203G;S2P, M71C, E96V, T213A; S2P, M71C, E96V, V225I; S2P, M71C, E96V, K237E;S2P, M71C, E96V, V256I; S2P, M71C, E96V, T258A; S2P, M71C, E96V, D264G;S2P, M71C, E96V, T283A; S2P, M71C, E96V, L289P; S2P, M71C, E96V, F312L;S2P, M71C, E96V, T348A; S2P, M71C, E96V, L380F; S2P, M71C, E96V, T394M;S2P, M71C, E96V, F396Y; S2P, M71C, E96V, R400C; S2P, M71C, E96V, T413A;S2P, M71C, E96V, E423D; S2P, M71C, E96V, L453P; S2P, M71C, E96V, F458S;S2P, M71C, E96V, E461G; S2P, M71C, E96V, F465C; S2P, M71C, E96V, N467T;S2P, M71C, E96V, N470S; S2P, M71C, E96V, A476T; S2P, M71C, E96V, Q494R;S2P, M71C, E96V, T496R; S2P, M71C, E96V, G504S; S2P, M71V, D101G, G112S;S2P, M71V, D101G, L114P; S2P, M71V, D101G, S130L; S2P, M71V, D101G,K145M; S2P, M71V, D101G, N166S; S2P, M71V, D101G, F171I; S2P, M71V,D101G, D203A; S2P, M71V, D101G, D203G; S2P, M71V, D101G, T213A; S2P,M71V, D101G, V225I; S2P, M71V, D101G, 237E; S2P, M71V, D101G, V256I;S2P, M71V, D101G, T258A; S2P, M71V, D101G, D264G; S2P, M71V, D101G,T283A; S2P, M71V, D101G, L289P; S2P, M71V, D101G, F312L; S2P, M71V,D101G, T348A; S2P, M71V, D101G, L380F; S2P, M71V, D101G, T394M; S2P,M71V, D101G, F396Y; S2P, M71V, D101G, R400C; S2P, M71V, D101G, T413A;S2P, M71V, D101G, E423D; S2P, M71V, D101G, L453P; S2P, M71V, D101G,F458S; S2P, M71V, D101G, E461G; S2P, M71V, D101G, F465C; S2P, M71V,D101G, N467T; S2P, M71V, D101G, N470S; S2P, M71V, D101G, A476T; S2P,M71V, D101G, Q494R; S2P, M71V, D101G, T496R; S2P, M71V, D101G, G504S;S2P, M71T, D101G, G112S; S2P, M71T, D101G, L114P; S2P, M71T, D101G,S130L; S2P, M71T, D101G, K145M; S2P, M71T, D101G, N166S; S2P, M71T,D101G, F171I; S2P, M71T, D101G, D203A; S2P, M71T, D101G, D203G; S2P,M71T, D101G, T213A; S2P, M71T, D101G, V225I; S2P, M71T, D101G, K237E;S2P, M71T, D101G, V256I; S2P, M71T, D101G, T258A; S2P, M71T, D101G,D264G; S2P, M71T, D101G, T283A; S2P, M71T, D101G, L289P; S2P, M71T,D101G, F312L; S2P, M71T, D101G, T348A; S2P, M71T, D101G, L380F; S2P,M71T, D101G, T394M; S2P, M71T, D101G, F396Y; S2P, M71T, D101G, R400C;S2P, M71T, D101G, T413A; S2P, M71T, D101G, E423D; S2P, M71T, D101G,L453P; S2P, M71T, D101G, F458S; S2P, M71T, D101G, E461G; S2P, M71T,D101G, F465C; S2P, M71T, D101G, N467T; S2P, M71T, D101G, N470S; S2P,M71T, D101G, A476T; S2P, M71T, D101G, Q494R; S2P, M71T, D101G, T496R;S2P, M71T, D101G, G504S; S2P, M71A, D101G, G112S; S2P, M71A, D101G,L114P; S2P, M71A, D101G, S130L; S2P, M71A, D101G, K145M; S2P, M71A,D101G, N166S; S2P, M71A, D101G, F171I; S2P, M71A, D101G, D203A; S2P,M71A, D101G, D203G; S2P, M71A, D101G, T213A; S2P, M71A, D101G, V225I;S2P, M71A, D101G, K237E; S2P, M71A, D101G, V256I; S2P, M71A, D101G,T258A; S2P, M71A, D101G, D264G; S2P, M71A, D101G, T283A; S2P, M71A,D101G, L289P; S2P, M71A, D101G, F312L; S2P, M71A, D101G, T348A; S2P,M71A, D101G, L380F; S2P, M71A, D101G, T394M; S2P, M71A, D101G, F396Y;S2P, M71A, D101G, R400C; S2P, M71A, D101G, T413A; S2P, M71A, D101G,E423D; S2P, M71A, D101G, L453P; S2P, M71A, D101G, F458S; S2P, M71A,D101G, E461G; S2P, M71A, D101G, F465C; S2P, M71A, D101G, N467T; S2P,M71A, D101G, N470S; S2P, M71A, D101G, A476T; S2P, M71A, D101G, Q494R;S2P, M71A, D101G, T496R; S2P, M71A, D101G, G504S; S2P, M71C, D101G,G112S; S2P, M71C, D101G, L114P; S2P, M71C, D101G, S130L; S2P, M71C,D101G, K145M; S2P, M71C, D101G, N166S; S2P, M71C, D101G, F171I; S2P,M71C, D101G, D203A; S2P, M71C, D101G, D203G; S2P, M71C, D101G, T213A;S2P, M71C, D101G, V225I; S2P, M71C, D101G, K237E; S2P, M71C, D101G,V256I; S2P, M71C, D101G, T258A; S2P, M71C, D101G, D264G; S2P, M71C,D101G, T283A; S2P, M71C, D101G, L289P; S2P, M71C, D101G, F312L; S2P,M71C, D101G, T348A; S2P, M71C, D101G, L380F; S2P, M71C, D101G, T394M;S2P, M71C, D101G, F396Y; S2P, M71C, D101G, R400C; S2P, M71C, D101G,T413A; S2P, M71C, D101G, E423D; S2P, M71C, D101G, L453P; S2P, M71C,D101G, F458S; S2P, M71C, D101G, E461G; S2P, M71C, D101G, F465C; S2P,M71C, D101G, N467T; S2P, M71C, D101G, N470S; S2P, M71C, D101G, A476T;S2P, M71C, D101G, Q494R; S2P, M71C, D101G, T496R; S2P, M71C, D101G,G504S; S2P, M71V, G112S, L114P; S2P, M71V, G112S, S130L; S2P, M71V,G112S, K145M; S2P, M71V, G112S, N166S; S2P, M71V, G112S, F171I; S2P,M71V, G112S, D203A; S2P, M71V, G112S, D203G; S2P, M71V, G112S, T213A;S2P, M71V, G112S, V225I; S2P, M71V, G112S, K237E; S2P, M71V, G112S,V256I; S2P, M71V, G112S, T258A; S2P, M71V, G112S, D264G; S2P, M71V,G112S, T283A; S2P, M71V, G112S, L289P; S2P, M71V, G112S, F312L; S2P,M71V, G112S, T348A; S2P, M71V, G112S, L380F; S2P, M71V, G112S, T394M;S2P, M71V, G112S, F396Y; S2P, M71V, G112S, R400C; S2P, M71V, G112S,T413A; S2P, M71V, G112S, E423D; S2P, M71V, G112S, L453P; S2P, M71V,G112S, F458S; S2P, M71V, G112S, E461G; S2P, M71V, G112S, F465C; S2P,M71V, G112S, N467T; S2P, M71V, G112S, N470S; S2P, M71V, G112S, A476T;S2P, M71V, G112S, Q494R; S2P, M71V, G112S, T496R; S2P, M71V, G112S,G504S; S2P, M71T, G112S, L114P; S2P, M71T, G112S, S130L; S2P, M71T,G112S, K145M; S2P, M71T, G112S, N166S; S2P, M71T, G112S, F171I; S2P,M71T, G112S, D203A; S2P, M71T, G112S, D203G; S2P, M71T, G112S, T213A;S2P, M71T, G112S, V225I; S2P, M71T, G112S, K237E; S2P, M71T, G112S,V256I; S2P, M71T, G112S, T258A; S2P, M71T, G112S, D264G; S2P, M71T,G112S, T283A; S2P, M71T, G112S, L289P; S2P, M71T, G112S, F312L; S2P,M71T, G112S, T348A; S2P, M71T, G112S, L380F; S2P, M71T, G112S, T394M;S2P, M71T, G112S, F396Y; S2P, M71T, G112S, R400C; S2P, M71T, G112S,T413A; S2P, M71T, G112S, E423D; S2P, M71T, G112S, L453P; S2P, M71T,G112S, F458S; S2P, M71T, G112S, E461G; S2P, M71T, G112S, F465C; S2P,M71T, G112S, N467T; S2P, M71T, G112S, N470S; S2P, M71T, G112S, A476T;S2P, M71T, G112S, Q494R; S2P, M71T, G112S, T496R; S2P, M71T, G112S,G504S; S2P, M71A, G112S, L114P; S2P, M71A, G112S, S130L; S2P, M71A,G112S, K145M; S2P, M71A, G112S, N166S; S2P, M71A, G112S, F171I; S2P,M71A, G112S, D203A; S2P, M71A, G112S, D203G; S2P, M71A, G112S, T213A;S2P, M71A, G112S, V225I; S2P, M71A, G112S, K237E; S2P, M71A, G112S,V256I; S2P, M71A, G112S, T258A; S2P, M71A, G112S, D264G; S2P, M71A,G112S, T283A; S2P, M71A, G112S, L289P; S2P, M71A, G112S, F312L; S2P,M71A, G112S, T348A; S2P, M71A, G112S, L380F; S2P, M71A, G112S, T394M;S2P, M71A, G112S, F396Y; S2P, M71A, G112S, R400C; S2P, M71A, G112S,T413A; S2P, M71A, G112S, E423D; S2P, M71A, G112S, L453P; S2P, M71A,G112S, F458S; S2P, M71A, G112S, E461G; S2P, M71A, G112S, F465C; S2P,M71A, G112S, N467T; S2P, M71A, G112S, N470S; S2P, M71A, G112S, A476T;S2P, M71A, G112S, Q494R; S2P, M71A, G112S, T496R; S2P, M71A, G112S,G504S; S2P, M71C, G112S, L114P; S2P, M71C, G112S, S130L; S2P, M71C,G112S, K145M; S2P, M71C, G112S, N166S; S2P, M71C, G112S, F171I; S2P,M71C, G112S, D203A; S2P, M71C, G112S, D203G; S2P, M71C, G112S, T213A;S2P, M71C, G112S, V225I; S2P, M71C, G112S, K237E; S2P, M71C, G112S,V256I; S2P, M71C, G112S, T258A; S2P, M71C, G112S, D264G; S2P, M71C,G112S, T283A; S2P, M71C, G112S, L289P; S2P, M71C, G112S, F312L; S2P,M71C, G112S, T348A; S2P, M71C, G112S, L380F; S2P, M71C, G112S, T394M;S2P, M71C, G112S, F396Y; S2P, M71C, G112S, R400C; S2P, M71C, G112S,T413A; S2P, M71C, G112S, E423D; S2P, M71C, G112S, L453P; S2P, M71C,G112S, F458S; S2P, M71C, G112S, E461G; S2P, M71C, G112S, F465C; S2P,M71C, G112S, N467T; S2P, M71C, G112S, N470S; S2P, M71C, G112S, A476T;S2P, M71C, G112S, Q494R; S2P, M71C, G112S, T496R; S2P, M71C, G112S,G504S; S2P, M71V, L114P, S130L; S2P, M71V, L114P, K145M; S2P, M71V,L114P, N166S; S2P, M71V, L114P, F171I; S2P, M71V, L114P, D203A; S2P,M71V, L114P, D203G; S2P, M71V, L114P, T213A; S2P, M71V, L114P, V225I;S2P, M71V, L114P, K237E; S2P, M71V, L114P, V256I; S2P, M71V, L114P,T258A; S2P, M71V, L114P, D264G; S2P, M71V, L114P, T283A; S2P, M71V,L114P, L289P; S2P, M71V, L114P, F312L; S2P, M71V, L114P, T348A; S2P,M71V, L114P, L380F; S2P, M71V, L114P, T394M; S2P, M71V, L114P, F396Y;S2P, M71V, L114P, R400C; S2P, M71V, L114P, T413A; S2P, M71V, L114P,E423D; S2P, M71V, L114P, L453P; S2P, M71V, L114P, F458S; S2P, M71V,L114P, E461G; S2P, M71V, L114P, F465C; S2P, M71V, L114P, N467T; S2P,M71V, L114P, N470S; S2P, M71V, L114P, A476T; S2P, M71V, L114P, Q494R;S2P, M71V, L114P, T496R; S2P, M71V, L114P, G504S; S2P, M71T, L114P,S130L; S2P, M71T, L114P, K145M; S2P, M71T, L114P, N166S; S2P, M71T,L114P, F171I; S2P, M71T, L114P, D203A; S2P, M71T, L114P, D203G; S2P,M71T, L114P, T213A; S2P, M71T, L114P, V225I; S2P, M71T, L114P, K237E;S2P, M71T, L114P, V256I; S2P, M71T, L114P, T258A; S2P, M71T, L114P,D264G; S2P, M71T, L114P, T283A; S2P, M71T, L114P, L289P; S2P, M71T,L114P, F312L; S2P, M71T, L114P, T348A; S2P, M71T, L114P, L380F; S2P,M71T, L114P, T394M; S2P, M71T, L114P, F396Y; S2P, M71T, L114P, R400C;S2P, M71T, L114P, T413A; S2P, M71T, L114P, E423D; S2P, M71T, L114P,L453P; S2P, M71T, L114P, F458S; S2P, M71T, L114P, E461G; S2P, M71T,L114P, F465C; S2P, M71T, L114P, N467T; S2P, M71T, L114P, N470S; S2P,M71T, L114P, A476T; S2P, M71T, L114P, Q494R; S2P, M71T, L114P, T496R;S2P, M71T, L114P, G504S; S2P, M71A, L114P, S130L; S2P, M71A, L114P,K145M; S2P, M71A, L114P, N166S; S2P, M71A, L114P, F171I; S2P, M71A,L114P, D203A; S2P, M71A, L114P, D203G; S2P, M71A, L114P, T213A; S2P,M71A, L114P, V225I; S2P, M71A, L114P, K237E; S2P, M71A, L114P, V256I;S2P, M71A, L114P, T258A; S2P, M71A, L114P, D264G; S2P, M71A, L114P,T283A; S2P, M71A, L114P, L289P; S2P, M71A, L114P, F312L; S2P, M71A,L114P, T348A; S2P, M71A, L114P, L380F; S2P, M71A, L114P, T394M; S2P,M71A, L114P, F396Y; S2P, M71A, L114P, R400C; S2P, M71A, L114P, T413A;S2P, M71A, L114P, E423D; S2P, M71A, L114P, L453P; S2P, M71A, L114P,F458S; S2P, M71A, L114P, E461G; S2P, M71A, L114P, F465C; S2P, M71A,L114P, N467T; S2P, M71A, L114P, N470S; S2P, M71A, L114P, A476T; S2P,M71A, L114P, Q494R; S2P, M71A, L114P, T496R; S2P, M71A, L114P, G504S;S2P, M71C, L114P, S130L; S2P, M71C, L114P, K145M; S2P, M71C, L114P,N166S; S2P, M71C, L114P, F171I; S2P, M71C, L114P, D203A; S2P, M71C,L114P, D203G; S2P, M71C, L114P, T213A; S2P, M71C, L114P, V225I; S2P,M71C, L114P, K237E; S2P, M71C, L114P, V256I; S2P, M71C, L114P, T258A;S2P, M71C, L114P, D264G; S2P, M71C, L114P, T283A; S2P, M71C, L114P,L289P; S2P, M71C, L114P, F312L; S2P, M71C, L114P, T348A; S2P, M71C,L114P, L380F; S2P, M71C, L114P, T394M; S2P, M71C, L114P, F396Y; S2P,M71C, L114P, R400C; S2P, M71C, L114P, T413A; S2P, M71C, L114P, E423D;S2P, M71C, L114P, L453P; S2P, M71C, L114P, F458S; S2P, M71C, L114P,E461G; S2P, M71C, L114P, F465C; S2P, M71C, L114P, N467T; S2P, M71C,L114P, N470S; S2P, M71C, L114P, A476T; S2P, M71C, L114P, Q494R; S2P,M71C, L114P, T496R; S2P, M71C, L114P, G504S; S2P, M71V, S130L, K145M;S2P, M71V, S130L, N166S; S2P, M71V, S130L, F171I; S2P, M71V, S130L,D203A; S2P, M71V, S130L, D203G; S2P, M71V, S130L, T213A; S2P, M71V,S130L, V225I; S2P, M71V, S130L, K237E; S2P, M71V, S130L, V256I; S2P,M71V, S130L, T258A; S2P, M71V, S130L, D264G; S2P, M71V, S130L, T283A;S2P, M71V, S130L, L289P; S2P, M71V, S130L, F312L; S2P, M71V, S130L,T348A; S2P, M71V, S130L, L380F; S2P, M71V, S130L, T394M; S2P, M71V,S130L, F396Y; S2P, M71V, S130L, R400C; S2P, M71V, S130L, T413A; S2P,M71V, S130L, E423D; S2P, M71V, S130L, L453P; S2P, M71V, S130L, F458S;S2P, M71V, S130L, E461G; S2P, M71V, S130L, F465C; S2P, M71V, S130L,N467T; S2P, M71V, S130L, N470S; S2P, M71V, S130L, A476T; S2P, M71V,S130L, Q494R; S2P, M71V, S130L, T496R; S2P, M71V, S130L, G504S; S2P,M71T, S130L, K145M; S2P, M71T, S130L, N166S; S2P, M71T, S130L, F171I;S2P, M71T, S130L, D203A; S2P, M71T, S130L, D203G; S2P, M71T, S130L,T213A; S2P, M71T, S130L, V225I; S2P, M71T, S130L, K237E; S2P, M71T,S130L, V256I; S2P, M71T, S130L, T258A; S2P, M71T, S130L, D264G; S2P,M71T, S130L, T283A; S2P, M71T, S130L, L289P; S2P, M71T, S130L, F312L;S2P, M71T, S130L, T348A; S2P, M71T, S130L, L380F; S2P, M71T, S130L,T394M; S2P, M71T, S130L, F396Y; S2P, M71T, S130L, R400C; S2P, M71T,S130L, T413A; S2P, M71T, S130L, E423D; S2P, M71T, S130L, L453P; S2P,M71T, S130L, F458S; S2P, M71T, S130L, E461G; S2P, M71T, S130L, F465C;S2P, M71T, S130L, N467T; S2P, M71T, S130L, N470S; S2P, M71T, S130L,A476T; S2P, M71T, S130L, Q494R; S2P, M71T, S130L, T496R; S2P, M71T,S130L, G504S; S2P, M71A, S130L, K145M; S2P, M71A, S130L, N166S; S2P,M71A, S130L, F171I; S2P, M71A, S130L, D203A; S2P, M71A, S130L, D203G;S2P, M71A, S130L, T213A; S2P, M71A, S130L, V225I; S2P, M71A, S130L,K237E; S2P, M71A, S130L, V256I; S2P, M71A, S130L, T258A; S2P, M71A,S130L, D264G; S2P, M71A, S130L, T283A; S2P, M71A, S130L, L289P; S2P,M71A, S130L, F312L; S2P, M71A, S130L, T348A; S2P, M71A, S130L, L380F;S2P, M71A, S130L, T394M; S2P, M71A, S130L, F396Y; S2P, M71A, S130L,R400C; S2P, M71A, S130L, T413A; S2P, M71A, S130L, E423D; S2P, M71A,S130L, L453P; S2P, M71A, S130L, F458S; S2P, M71A, S130L, E461G; S2P,M71A, S130L, F465C; S2P, M71A, S130L, N467T; S2P, M71A, S130L, N470S;S2P, M71A, S130L, A476T; S2P, M71A, S130L, Q494R; S2P, M71A, S130L,T496R; S2P, M71A, S130L, G504S; S2P, M71C, S130L, K145M; S2P, M71C,S130L, N166S; S2P, M71C, S130L, F171I; S2P, M71C, S130L, D203A; S2P,M71C, S130L, D203G; S2P, M71C, S130L, T213A; S2P, M71C, S130L, V225I;S2P, M71C, S130L, K237E; S2P, M71C, S130L, V256I; S2P, M71C, S130L,T258A; S2P, M71C, S130L, D264G; S2P, M71C, S130L, T283A; S2P, M71C,S130L, L289P; S2P, M71C, S130L, F312L; S2P, M71C, S130L, T348A; S2P,M71C, S130L, L380F; S2P, M71C, S130L, T394M; S2P, M71C, S130L, F396Y;S2P, M71C, S130L, R400C; S2P, M71C, S130L, T413A; S2P, M71C, S130L,E423D; S2P, M71C, S130L, L453P; S2P, M71C, S130L, F458S; S2P, M71C,S130L, E461G; S2P, M71C, S130L, F465C; S2P, M71C, S130L, N467T; S2P,M71C, S130L, N470S; S2P, M71C, S130L, A476T; S2P, M71C, S130L, Q494R;S2P, M71C, S130L, T496R; S2P, M71C, S130L, G504S; S2P, M71V, K145M,N166S; S2P, M71V, K145M, F171I; S2P, M71V, K145M, D203A; S2P, M71V,K145M, D203G; S2P, M71V, K145M, T213A; S2P, M71V, K145M, V225I; S2P,M71V, K145M, K237E; S2P, M71V, K145M, V256I; S2P, M71V, K145M, T258A;S2P, M71V, K145M, D264G; S2P, M71V, K145M, T283A; S2P, M71V, K145M,L289P; S2P, M71V, K145M, F312L; S2P, M71V, K145M, T348A; S2P, M71V,K145M, L380F; S2P, M71V, K145M, T394M; S2P, M71V, K145M, F396Y; S2P,M71V, K145M, R400C; S2P, M71V, K145M, T413A; S2P, M71V, K145M, E423D;S2P, M71V, K145M, L453P; S2P, M71V, K145M, F458S; S2P, M71V, K145M,E461G; S2P, M71V, K145M, F465C; S2P, M71V, K145M, N467T; S2P, M71V,K145M, N470S; S2P, M71V, K145M, A476T; S2P, M71V, K145M, Q494R; S2P,M71V, K145M, T496R; S2P, M71V, K145M, G504S; S2P, M71T, K145M, N166S;S2P, M71T, K145M, F171I; S2P, M71T, K145M, D203A; S2P, M71T, K145M,D203G; S2P, M71T, K145M, T213A; S2P, M71T, K145M, V225I; S2P, M71T,K145M, K237E; S2P, M71T, K145M, V256I; S2P, M71T, K145M, T258A; S2P,M71T, K145M, D264G; S2P, M71T, K145M, T283A; S2P, M71T, K145M, L289P;S2P, M71T, K145M, F312L; S2P, M71T, K145M, T348A; S2P, M71T, K145M,L380F; S2P, M71T, K145M, T394M; S2P, M71T, K145M, F396Y; S2P, M71T,K145M, R400C; S2P, M71T, K145M, T413A; S2P, M71T, K145M, E423D; S2P,M71T, K145M, L453P; S2P, M71T, K145M, F458S; S2P, M71T, K145M, E461G;S2P, M71T, K145M, F465C; S2P, M71T, K145M, N467T; S2P, M71T, K145M,N470S; S2P, M71T, K145M, A476T; S2P, M71T, K145M, Q494R; S2P, M71T,K145M, T496R; S2P, M71T, K145M, G504S; S2P, M71A, K145M, N166S; S2P,M71A, K145M, F171I; S2P, M71A, K145M, D203A; S2P, M71A, K145M, D203G;S2P, M71A, K145M, T213A; S2P, M71A, K145M, V225I; S2P, M71A, K145M,K237E; S2P, M71A, K145M, V256I; S2P, M71A, K145M, T258A; S2P, M71A,K145M, D264G; S2P, M71A, K145M, T283A; S2P, M71A, K145M, L289P; S2P,M71A, K145M, F312L; S2P, M71A, K145M, T348A; S2P, M71A, K145M, L380F;S2P, M71A, K145M, T394M; S2P, M71A, K145M, F396Y; S2P, M71A, K145M,R400C; S2P, M71A, K145M, T413A; S2P, M71A, K145M, E423D; S2P, M71A,K145M, L453P; S2P, M71A, K145M, F458S; S2P, M71A, K145M, E461G; S2P,M71A, K145M, F465C; S2P, M71A, K145M, N467T; S2P, M71A, K145M, N470S;S2P, M71A, K145M, A476T; S2P, M71A, K145M, Q494R; S2P, M71A, K145M,T496R; S2P, M71A, K145M, G504S; S2P, M71C, K145M, N166S; S2P, M71C,K145M, F171I; S2P, M71C, K145M, D203A; S2P, M71C, K145M, D203G; S2P,M71C, K145M, T213A; S2P, M71C, K145M, V225I, S2P, M71C, K145M, K237E,S2P, M71C, K145M, V256I, S2P, M71C, K145M, T258A, S2P, M71C, K145M,D264G, S2P, M71C, K145M, T283A; S2P, M71C, K145M, L289P; S2P, M71C,K145M, F312L; S2P, M71C, K145M, T348A; S2P, M71C, K145M, L380F; S2P,M71C, K145M, T394M; S2P, M71C, K145M, F396Y; S2P, M71C, K145M, R400C;S2P, M71C, K145M, T413A; S2P, M71C, K145M, E423D; S2P, M71C, K145M,L453P; S2P, M71C, K145M, F458S; S2P, M71C, K145M, E461G; S2P, M71C,K145M, F465C; S2P, M71C, K145M, N467T; S2P, M71C, K145M, N470S; S2P,M71C, K145M, A476T; S2P, M71C, K145M, Q494R; S2P, M71C, K145M, T496R;S2P, M71C, K145M, G504S; S2P, M71V, D203A, N166S; S2P, M71V, D203A,F171I; S2P, M71V, D203A, T213A; S2P, M71V, D203A, V225I; S2P, M71V,D203A, K237E; S2P, M71V, D203A, V256I; S2P, M71V, D203A, T258A; S2P,M71V, D203A, D264G; S2P, M71V, D203A, T283A; S2P, M71V, D203A, L289P;S2P, M71V, D203A, F312L; S2P, M71V, D203A, T348A; S2P, M71V, D203A,L380F; S2P, M71V, D203A, T394M; S2P, M71V, D203A, F396Y; S2P, M71V,D203A, R400C; S2P, M71V, D203A, T413A; S2P, M71V, D203A, E423D; S2P,M71V, D203A, L453P; S2P, M71V, D203A, F458S; S2P, M71V, D203A, E461G;S2P, M71V, D203A, F465C; S2P, M71V, D203A, N467T; S2P, M71V, D203A,N470S; S2P, M71V, D203A, A476T; S2P, M71V, D203A, Q494R; S2P, M71V,D203A, T496R; S2P, M71V, D203A, G504S; S2P, M71T, D203A, N166S; S2P,M71T, D203A, F171I; S2P, M71T, D203A, T213A; S2P, M71T, D203A, V225I;S2P, M71T, D203A, K237E; S2P, M71T, D203A, V256I; S2P, M71T, D203A,T258A; S2P, M71T, D203A, D264G; S2P, M71T, D203A, T283A; S2P, M71T,D203A, L289P; S2P, M71T, D203A, F312L; S2P, M71T, D203A, T348A; S2P,M71T, D203A, L380F; S2P, M71T, D203A, T394M; S2P, M71T, D203A, F396Y;S2P, M71T, D203A, R400C; S2P, M71T, D203A, T413A; S2P, M71T, D203A,E423D; S2P, M71T, D203A, L453P; S2P, M71T, D203A, F458S; S2P, M71T,D203A, E461G; S2P, M71T, D203A, F465C; S2P, M71T, D203A, N467T; S2P,M71T, D203A, N470S; S2P, M71T, D203A, A476T; S2P, M71T, D203A, Q494R;S2P, M71T, D203A, T496R; S2P, M71T, D203A, G504S; S2P, M71A, D203A,N166S; S2P, M71A, D203A, T213A; S2P, M71A, D203A, V225I; S2P, M71A,D203A, K237E; S2P, M71A, D203A, V256I; S2P, M71A, D203A, T258A; S2P,M71A, D203A, D264G; S2P, M71A, D203A, T283A; S2P, M71A, D203A, L289P;S2P, M71A, D203A, F312L; S2P, M71A, D203A, T348A; S2P, M71A, D203A,L380F; S2P, M71A, D203A, T394M; S2P, M71A, D203A, F396Y; S2P, M71A,D203A, R400C; S2P, M71A, D203A, T413A; S2P, M71A, D203A, E423D; S2P,M71A, D203A, L453P; S2P, M71A, D203A, F458S; S2P, M71A, D203A, E461G;S2P, M71A, D203A, F465C; S2P, M71A, D203A, N467T; S2P, M71A, D203A,N470S; S2P, M71A, D203A, A476T; S2P, M71A, D203A, Q494R; S2P, M71A,D203A, T496R; S2P, M71A, D203A, G504S; S2P, M71C, D203A, N166S; S2P,M71C, D203A, F171I; S2P, M71C, D203A, T213A; S2P, M71C, D203A, V225I;S2P, M71C, D203A, K237E; S2P, M71C, D203A, V256I; S2P, M71C, D203A,T258A; S2P, M71C, D203A, D264G; S2P, M71C, D203A, T283A; S2P, M71C,D203A, L289P; S2P, M71C, D203A, F312L; S2P, M71C, D203A, T348A; 2P,M71C, D203A, L380F; S2P, M71C, D203A, T394M; S2P, M71C, D203A, F396Y;S2P, M71C, D203A, R400C; S2P, M71C, D203A, T413A; S2P, M71C, D203A,E423D; S2P, M71C, D203A, L453P; S2P, M71C, D203A, F458S; S2P, M71C,D203A, E461G; S2P, M71C, D203A, F465C; S2P, M71C, D203A, N467T; S2P,M71C, D203A, N470S; S2P, M71C, D203A, A476T; S2P, M71C, D203A, Q494R;S2P, M71C, D203A, T496R; S2P, M71C, D203A, G504S; S2P, M71V, D203C,N166S; S2P, M71V, D203C, F171I; S2P, M71V, D203C, T213A; S2P, M71V,D203C, V225I; S2P, M71V, D203C, K237E; S2P, M71V, D203C, V256I; S2P,M71V, D203C, T258A; S2P, M71V, D203C, D264G; S2P, M71V, D203C, T283A;S2P, M71V, D203C, L289P; S2P, M71V, D203C, F312L; S2P, M71V, D203C,T348A; S2P, M71V, D203C, L380F; S2P, M71V, D203C, T394M; S2P, M71V,D203C, F396Y; S2P, M71V, D203C, R400C; S2P, M71V, D203C, T413A; S2P,M71V, D203C, E423D; S2P, M71V, D203C, L453P; S2P, M71V, D203C, F458S;2P, M71V, D203C, E461G; S2P, M71V, D203C, F465C; S2P, M71V, D203C,N467T; S2P, M71V, D203C, N470S; S2P, M71V, D203C, A476T; S2P, M71V,D203C, Q494R; S2P, M71V, D203C, T496R; S2P, M71V, D203C, G504S; S2P,M71T, D203C, N166S; S2P, M71T, D203C, F171I; S2P, M71T, D203C, T213A;S2P, M71T, D203C, V225I; S2P, M71T, D203C, K237E; S2P, M71T, D203C,V256I; S2P, M71T, D203C, T258A; S2P, M71T, D203C, D264G; S2P, M71T,D203C, T283A; S2P, M71T, D203C, L289P; S2P, M71T, D203C, F312L; S2P,M71T, D203C, T348A; S2P, M71T, D203C, L380F; S2P, M71T, D203C, T394M;S2P, M71T, D203C, F396Y; S2P, M71T, D203C, R400C; S2P, M71T, D203C,T413A; S2P, M71T, D203C, E423D; S2P, M71T, D203C, L453P; S2P, M71T,D203C, F458S; S2P, M71T, D203C, E461G; S2P, M71T, D203C, F465C; S2P,M71T, D203C, N467T; S2P, M71T, D203C, N470S; S2P, M71T, D203C, A476T;S2P, M71T, D203C, Q494R; S2P, M71T, D203C, T496R; S2P, M71T, D203C,G504S; S2P, M71A, D203C, N166S; S2P, M71A, D203C, T213A; S2P, M71A,D203C, V225I; S2P, M71A, D203C, K237E; S2P, M71A, D203C, V256I; S2P,M71A, D203C, T258A; S2P, M71A, D203C, D264G; S2P, M71A, D203C, T283A;S2P, M71A, D203C, L289P; S2P, M71A, D203C, F312L; S2P, M71A, D203C,T348A; S2P, M71A, D203C, L380F; S2P, M71A, D203C, T394M; S2P, M71A,D203C, F396Y; S2P, M71A, D203C, R400C; S2P, M71A, D203C, T413A; S2P,M71A, D203C, E423D; S2P, M71A, D203C, L453P; S2P, M71A, D203C, F458S;S2P, M71A, D203C, E461G; S2P, M71A, D203C, F465C; S2P, M71A, D203C,N467T; S2P, M71A, D203C, N470S; S2P, M71A, D203C, A476T; S2P, M71A,D203C, Q494R; S2P, M71A, D203C, T496R; S2P, M71A, D203C, G504S; S2P,M71C, D203C, N166S; S2P, M71C, D203C, F171I; S2P, M71C, D203C, T213A;S2P, M71C, D203C, V225I; S2P, M71C, D203C, K237E; S2P, M71C, D203C,V256I; S2P, M71C, D203C, T258A; S2P, M71C, D203C, D264G; S2P, M71C,D203C, T283A; S2P, M71C, D203C, L289P; S2P, M71C, D203C, F312L; S2P,M71C, D203C, T348A; S2P, M71C, D203C, L380F; S2P, M71C, D203C, T394M;S2P, M71C, D203C, F396Y; S2P, M71C, D203C, R400C; S2P, M71C, D203C,T413A; S2P, M71C, D203C, E423D; S2P, M71C, D203C, L453P; S2P, M71C,D203C, F458S; S2P, M71C, D203C, E461G; S2P, M71C, D203C, F465C; S2P,M71C, D203C, N467T; S2P, M71C, D203C, N470S; S2P, M71C, D203C, A476T;S2P, M71C, D203C, Q494R; S2P, M71C, D203C, T496R; S2P, M71C, D203C,G504S; S2P, M71V, T213A, V225I; S2P, M71V, T213A, K237E; S2P, M71V,T213A, V256I; S2P, M71V, T213A, T258A; S2P, M71V, T213A, D264G; S2P,M71V, T213A, T283A; S2P, M71V, T213A, L289P; S2P, M71V, T213A, F312L;S2P, M71V, T213A, T348A; S2P, M71V, T213A, L380F; S2P, M71V, T213A,T394M; S2P, M71V, T213A, F396Y; S2P, M71V, T213A, R400C; S2P, M71V,T213A, T413A; S2P, M71V, T213A, E423D; S2P, M71V, T213A, L453P; S2P,M71V, T213A, F458S; S2P, M71V, T213A, E461G; S2P, M71V, T213A, F465C;S2P, M71V, T213A, N467T; S2P, M71V, T213A, N470S; S2P, M71V, T213A,A476T; S2P, M71V, T213A, Q494R; S2P, M71V, T213A, T496R; S2P, M71V,T213A, G504S; S2P, M71T, T213A, N166S; S2P, M71T, T213A, F171I; S2P,M71T, T213A, V225I; S2P, M71T, T213A, K237E; S2P, M71T, T213A, V256I;S2P, M71T, T213A, T258A; S2P, M71T, T213A, D264G; S2P, M71T, T213A,T283A; S2P, M71T, T213A, L289P; S2P, M71T, T213A, F312L; S2P, M71T,T213A, T348A; S2P, M71T, T213A, L380F; S2P, M71T, T213A, T394M; S2P,M71T, T213A, F396Y; S2P, M71T, T213A, R400C; S2P, M71T, T213A, T413A;S2P, M71T, T213A, E423D; S2P, M71T, T213A, L453P; S2P, M71T, T213A,F458S; S2P, M71T, T213A, E461G; S2P, M71T, T213A, F465C; S2P, M71T,T213A, N467T; S2P, M71T, T213A, N470S; S2P, M71T, T213A, A476T; S2P,M71T, T213A, Q494R; S2P, M71T, T213A, T496R; S2P, M71T, T213A, G504S;S2P, M71A, T213A, N166S; S2P, M71A, T213A, V225I; S2P, M71A, T213A,K237E; S2P, M71A, T213A, V256I; S2P, M71A, T213A, T258A; S2P, M71A,T213A, D264G; S2P, M71A, T213A, T283A; S2P, M71A, T213A, L289P; S2P,M71A, T213A, F312L; S2P, M71A, T213A, T348A; S2P, M71A, T213A, L380F;S2P, M71A, T213A, T394M; S2P, M71A, T213A, F396Y; S2P, M71A, T213A,R400C; S2P, M71A, T213A, T413A; S2P, M71A, T213A, E423D; S2P, M71A,T213A, L453P; S2P, M71A, T213A, F458S; S2P, M71A, T213A, E461G; S2P,M71A, T213A, F465C; S2P, M71A, T213A, N467T; S2P, M71A, T213A, N470S;S2P, M71A, T213A, A476T; S2P, M71A, T213A, Q494R; S2P, M71A, T213A,T496R; S2P, M71A, T213A, G504S; S2P, M71C, T213A, N166S; S2P, M71C,T213A, F171I; S2P, M71C, T213A, V225I; S2P, M71C, T213A, K237E; S2P,M71C, T213A, V256I; S2P, M71C, T213A, T258A; S2P, M71C, T213A, D264G;S2P, M71C, T213A, T283A; S2P, M71C, T213A, L289P; S2P, M71C, T213A,F312L; S2P, M71C, T213A, T348A; S2P, M71C, T213A, L380F; S2P, M71C,T213A, T394M; S2P, M71C, T213A, F396Y; S2P, M71C, T213A, R400C; S2P,M71C, T213A, T413A; S2P, M71C, T213A, E423D; S2P, M71C, T213A, L453P;S2P, M71C, T213A, F458S; S2P, M71C, T213A, E461G; S2P, M71C, T213A,F465C; S2P, M71C, T213A, N467T; S2P, M71C, T213A, N470S; S2P, M71C,T213A, A476T; S2P, M71C, T213A, Q494R; S2P, M71C, T213A, T496R; S2P,M71C, T213A, G504S; S2P, M71V, V225I, K237E; S2P, M71V, V225I, V256I;S2P, M71V, V225I, T258A; S2P, M71V, V225I, D264G; S2P, M71V, V225I,T283A; S2P, M71V, V225I, L289P; S2P, M71V, V225I, F312L; S2P, M71V,V225I, T348A; S2P, M71V, V225I, L380F; S2P, M71V, V225I, T394M; S2P,M71V, V225I, F396Y; S2P, M71V, V225I, R400C; S2P, M71V, V225I, T413A;S2P, M71V, V225I, E423D; S2P, M71V, V225I, L453P; S2P, M71V, V225I,F458S; S2P, M71V, V225I, E461G; S2P, M71V, V225I, F465C; S2P, M71V,V225I, N467T; S2P, M71V, V225I, N470S; S2P, M71V, V225I, A476T; S2P,M71V, V225I, Q494R; S2P, M71V, V225I, T496R; S2P, M71V, V225I, G504S;S2P, M71T, V225I, N166S; S2P, M71T, V225I, F171I; S2P, M71T, V225I,K237E; S2P, M71T, V225I, V256I; S2P, M71T, V225I, T258A; S2P, M71T,V225I, D264G; S2P, M71T, V225I, T283A; S2P, M71T, V225I, L289P; S2P,M71T, V225I, F312L; S2P, M71T, V225I, T348A; S2P, M71T, V225I, L380F;S2P, M71T, V225I, T394M; S2P, M71T, V225I, F396Y; S2P, M71T, V225I,R400C; S2P, M71T, V225I, T413A; S2P, M71T, V225I, E423D; S2P, M71T,V225I, L453P; S2P, M71T, V225I, F458S; S2P, M71T, V225I, E461G; S2P,M71T, V225I, F465C; S2P, M71T, V225I, N467T; S2P, M71T, V225I, N470S;S2P, M71T, V225I, A476T; S2P, M71T, V225I, Q494R; S2P, M71T, V225I,T496R; S2P, M71T, V225I, G504S; S2P, M71A, V225I, N166S; S2P, M71A,V225I, K237E; S2P, M71A, V225I, V256I; S2P, M71A, V225I, T258A; S2P,M71A, V225I, D264G; S2P, M71A, V225I, T283A; S2P, M71A, V225I, L289P;S2P, M71A, V225I, F312L; S2P, M71A, V225I, T348A; S2P, M71A, V225I,L380F; S2P, M71A, V225I, T394M; S2P, M71A, V225I, F396Y; S2P, M71A,V225I, R400C; S2P, M71A, V225I, T413A; S2P, M71A, V225I, E423D; S2P,M71A, V225I, L453P; S2P, M71A, V225I, F458S; S2P, M71A, V225I, E461G;S2P, M71A, V225I, F465C; S2P, M71A, V225I, N467T; S2P, M71A, V225I,N470S; S2P, M71A, V225I, A476T; S2P, M71A, V225I, Q494R; S2P, M71A,V225I, T496R; S2P, M71A, V225I, G504S; S2P, M71C, V225I, N166S; S2P,M71C, V225I, F171I; S2P, M71C, V225I, K237E; S2P, M71C, V225I, V256I;S2P, M71C, V225I, T258A; S2P, M71C, V225I, D264G; S2P, M71C, V225I,T283A; S2P, M71C, V225I, L289P; S2P, M71C, V225I, F312L; S2P, M71C,V225I, T348A; S2P, M71C, V225I, L380F; S2P, M71C, V225I, T394M; S2P,M71C, V225I, F396Y; S2P, M71C, V225I, R400C; S2P, M71C, V225I, T413A;S2P, M71C, V225I, E423D; S2P, M71C, V225I, L453P; S2P, M71C, V225I,F458S; S2P, M71C, V225I, E461G; S2P, M71C, V225I, F465C; S2P, M71C,V225I, N467T; S2P, M71C, V225I, N470S; S2P, M71C, V225I, A476T; S2P,M71C, V225I, Q494R; S2P, M71C, V225I, T496R; S2P, M71C, V225I, G504S;S2P, M71V, K237E, V256I; S2P, M71V, K237E, T258A; S2P, M71V, K237E,D264G; S2P, M71V, K237E, T283A; S2P, M71V, K237E, L289P; S2P, M71V,K237E, F312L; S2P, M71V, K237E, T348A; S2P, M71V, K237E, L380F; S2P,M71V, K237E, T394M; S2P, M71V, K237E, F396Y; S2P, M71V, K237E, R400C;S2P, M71V, K237E, T413A; S2P, M71V, K237E, E423D; S2P, M71V, K237E,L453P; S2P, M71V, K237E, F458S; S2P, M71V, K237E, E461G; S2P, M71V,K237E, F465C; S2P, M71V, K237E, N467T; S2P, M71V, K237E, N470S; S2P,M71V, K237E, A476T; S2P, M71V, K237E, Q494R; S2P, M71V, K237E, T496R;S2P, M71V, K237E, G504S; S2P, M71T, K237E, V256I; S2P, M71T, K237E,T258A; S2P, M71T, K237E, D264G; S2P, M71T, K237E, T283A; S2P, M71T,K237E, L289P; S2P, M71T, K237E, F312L; S2P, M71T, K237E, T348A; S2P,M71T, K237E, L380F; S2P, M71T, K237E, T394M; S2P, M71T, K237E, F396Y;S2P, M71T, K237E, R400C; S2P, M71T, K237E, T413A; S2P, M71T, K237E,E423D; S2P, M71T, K237E, L453P; S2P, M71T, K237E, F458S; S2P, M71T,K237E, E461G; S2P, M71T, K237E, F465C; S2P, M71T, K237E, N467T; S2P,M71T, K237E, N470S; S2P, M71T, K237E, A476T; S2P, M71T, K237E, Q494R;S2P, M71T, K237E, T496R; S2P, M71T, K237E, G504S; S2P, M71A, K237E,V256I; S2P, M71A, K237E, T258A; S2P, M71A, K237E, D264G; S2P, M71A,K237E, T283A; S2P, M71A, K237E, L289P; S2P, M71A, K237E, F312L; S2P,M71A, K237E, T348A; S2P, M71A, K237E, L380F; S2P, M71A, K237E, T394M;S2P, M71A, K237E, F396Y; S2P, M71A, K237E, R400C; S2P, M71A, K237E,T413A; S2P, M71A, K237E, E423D; S2P, M71A, K237E, L453P; S2P, M71A,K237E, F458S; S2P, M71A, K237E, E461G; S2P, M71A, K237E, F465C; S2P,M71A, K237E, N467T; S2P, M71A, K237E, N470S; S2P, M71A, K237E, A476T;S2P, M71A, K237E, Q494R; S2P, M71A, K237E, T496R; S2P, M71A, K237E,G504S; S2P, M71C, K237E, V256I; S2P, M71C, K237E, T258A; S2P, M71C,K237E, D264G; S2P, M71C, K237E, T283A; S2P, M71C, K237E, L289P; S2P,M71C, K237E, F312L; S2P, M71C, K237E, T348A; S2P, M71C, K237E, L380F;S2P, M71C, K237E, T394M; S2P, M71C, K237E, F396Y; S2P, M71C, K237E,R400C; S2P, M71C, K237E, T413A; S2P, M71C, K237E, E423D; S2P, M71C,K237E, L453P; S2P, M71C, K237E, F458S; S2P, M71C, K237E, E461G; S2P,M71C, K237E, F465C; S2P, M71C, K237E, N467T; S2P, M71C, K237E, N470S;S2P, M71C, K237E, A476T; S2P, M71C, K237E, Q494R; S2P, M71C, K237E,T496R; S2P, M71C, K237E, G504S; S2P, M71V, V256I, T258A; S2P, M71V,V256I, D264G; S2P, M71V, V256I, T283A; S2P, M71V, V256I, L289P; S2P,M71V, V256I, F312L; S2P, M71V, V256I, T348A; S2P, M71V, V256I, L380F;S2P, M71V, V256I, T394M; S2P, M71V, V256I, F396Y; S2P, M71V, V256I,R400C; S2P, M71V, V256I, T413A; S2P, M71V, V256I, E423D; S2P, M71V,V256I, L453P; S2P, M71V, V256I, F458S; S2P, M71V, V256I, E461G; S2P,M71V, V256I, F465C; S2P, M71V, V256I, N467T; S2P, M71V, V256I, N470S;S2P, M71V, V256I, A476T; S2P, M71V, V256I, Q494R; S2P, M71V, V256I,T496R; S2P, M71V, V256I, G504S; S2P, M71T, V256I, T258A; S2P, M71T,V256I, D264G; S2P, M71T, V256I, T283A; S2P, M71T, V256I, L289P; S2P,M71T, V256I, F312L; S2P, M71T, V256I, T348A; S2P, M71T, V256I, L380F;S2P, M71T, V256I, T394M; S2P, M71T, V256I, F396Y; S2P, M71T, V256I,R400C; S2P, M71T, V256I, T413A; S2P, M71T, V256I, E423D; S2P, M71T,V256I, L453P; S2P, M71T, V256I, F458S; S2P, M71T, V256I, E461G; S2P,M71T, V256I, F465C; S2P, M71T, V256I, N467T; S2P, M71T, V256I, N470S;S2P, M71T, V256I, A476T; S2P, M71T, V256I, Q494R; S2P, M71T, V256I,T496R; S2P, M71T, V256I, G504S; S2P, M71A, V256I, T258A; S2P, M71A,V256I, D264G; S2P, M71A, V256I, T283A; S2P, M71A, V256I, L289P; S2P,M71A, V256I, F312L; S2P, M71A, V256I, T348A; S2P, M71A, V256I, L380F;S2P, M71A, V256I, T394M; S2P, M71A, V256I, F396Y; S2P, M71A, V256I,R400C; S2P, M71A, V256I, T413A; S2P, M71A, V256I, E423D; S2P, M71A,V256I, L453P; S2P, M71A, V256I, F458S; S2P, M71A, V256I, E461G; S2P,M71A, V256I, F465C; S2P, M71A, V256I, N467T; S2P, M71A, V256I, N470S;S2P, M71A, V256I, A476T; S2P, M71A, V256I, Q494R; S2P, M71A, V256I,T496R; S2P, M71A, V256I, G504S; S2P, M71C, V256I, T258A; S2P, M71C,V256I, D264G; S2P, M71C, V256I, T283A; S2P, M71C, V256I, L289P; S2P,M71C, V256I, F312L; S2P, M71C, V256I, T348A; S2P, M71C, V256I, L380F;S2P, M71C, V256I, T394M; S2P, M71C, V256I, F396Y; S2P, M71C, V256I,R400C; S2P, M71C, V256I, T413A; S2P, M71C, V256I, E423D; S2P, M71C,V256I, L453P; S2P, M71C, V256I, F458S; S2P, M71C, V256I, E461G; S2P,M71C, V256I, F465C; S2P, M71C, V256I, N467T; S2P, M71C, V256I, N470S;S2P, M71C, V256I, A476T; S2P, M71C, V256I, Q494R; S2P, M71C, V256I,T496R; S2P, M71C, V256I, G504S; S2P, M71V, T258A, D264G; S2P, M71V,T258A, T283A; S2P, M71V, T258A, L289P; S2P, M71V, T258A, F312L; S2P,M71V, T258A, T348A; S2P, M71V, T258A, L380F; S2P, M71V, T258A, T394M;S2P, M71V, T258A, F396Y; S2P, M71V, T258A, R400C; S2P, M71V, T258A,T413A; S2P, M71V, T258A, E423D; S2P, M71V, T258A, L453P; S2P, M71V,T258A, F458S; S2P, M71V, T258A, E461G; S2P, M71V, T258A, F465C; S2P,M71V, T258A, N467T; S2P, M71V, T258A, N470S; S2P, M71V, T258A, A476T;S2P, M71V, T258A, Q494R; S2P, M71V, T258A, T496R; S2P, M71V, T258A,G504S; S2P, M71T, T258A, D264G; S2P, M71T, T258A, T283A; S2P, M71T,T258A, L289P; S2P, M71T, T258A, F312L; S2P, M71T, T258A, T348A; S2P,M71T, T258A, L380F; S2P, M71T, T258A, T394M; S2P, M71T, T258A, F396Y;S2P, M71T, T258A, R400C; S2P, M71T, T258A, T413A; S2P, M71T, T258A,E423D; S2P, M71T, T258A, L453P; S2P, M71T, T258A, F458S; S2P, M71T,T258A, E461G; S2P, M71T, T258A, F465C; S2P, M71T, T258A, N467T; S2P,M71T, T258A, N470S; S2P, M71T, T258A, A476T; S2P, M71T, T258A, Q494R;S2P, M71T, T258A, T496R; S2P, M71T, T258A, G504S; S2P, M71A, T258A,D264G; S2P, M71A, T258A, T283A; S2P, M71A, T258A, L289P; S2P, M71A,T258A, F312L; S2P, M71A, T258A, T348A; S2P, M71A, T258A, L380F; S2P,M71A, T258A, T394M; S2P, M71A, T258A, F396Y; S2P, M71A, T258A, R400C;S2P, M71A, T258A, T413A; S2P, M71A, T258A, E423D; S2P, M71A, T258A,L453P; S2P, M71A, T258A, F458S; S2P, M71A, T258A, E461G; S2P, M71A,T258A, F465C; S2P, M71A, T258A, N467T; S2P, M71A, T258A, N470S; S2P,M71A, T258A, A476T; S2P, M71A, T258A, Q494R; S2P, M71A, T258A, T496R;S2P, M71A, T258A, G504S; S2P, M71C, T258A, D264G; S2P, M71C, T258A,T283A; S2P, M71C, T258A, L289P; S2P, M71C, T258A, F312L; S2P, M71C,T258A, T348A; S2P, M71C, T258A, L380F; S2P, M71C, T258A, T394M; S2P,M71C, T258A, F396Y; S2P, M71C, T258A, R400C; S2P, M71C, T258A, T413A;S2P, M71C, T258A, E423D; S2P, M71C, T258A, L453P; S2P, M71C, T258A,F458S; S2P, M71C, T258A, E461G; S2P, M71C, T258A, F465C; S2P, M71C,T258A, N467T; S2P, M71C, T258A, N470S; S2P, M71C, T258A, A476T; S2P,M71C, T258A, Q494R; S2P, M71C, T258A, T496R; S2P, M71C, T258A, G504S;S2P, M71V, D264G, T283A; S2P, M71V, D264G, L289P; S2P, M71V, D264G,F312L; S2P, M71V, D264G, T348A; S2P, M71V, D264G, L380F; S2P, M71V,D264G, T394M; S2P, M71V, D264G, F396Y; S2P, M71V, D264G, R400C; S2P,M71V, D264G, T413A; S2P, M71V, D264G, E423D; S2P, M71V, D264G, L453P;S2P, M71V, D264G, F458S; S2P, M71V, D264G, E461G; S2P, M71V, D264G,F465C; S2P, M71V, D264G, N467T; S2P, M71V, D264G, N470S; S2P, M71V,D264G, A476T; S2P, M71V, D264G, Q494R; S2P, M71V, D264G, T496R; S2P,M71V, D264G, G504S; S2P, M71T, D264G, T283A; S2P, M71T, D264G, L289P;S2P, M71T, D264G, F312L; S2P, M71T, D264G, T348A; S2P, M71T, D264G,L380F; S2P, M71T, D264G, T394M; S2P, M71T, D264G, F396Y; S2P, M71T,D264G, R400C; S2P, M71T, D264G, T413A; S2P, M71T, D264G, E423D; S2P,M71T, D264G, L453P; S2P, M71T, D264G, F458S; S2P, M71T, D264G, E461G;S2P, M71T, D264G, F465C; S2P, M71T, D264G, N467T; S2P, M71T, D264G,N470S; S2P, M71T, D264G, A476T; S2P, M71T, D264G, Q494R; S2P, M71T,D264G, T496R; S2P, M71T, D264G, G504S; S2P, M71A, D264G, T283A; S2P,M71A, D264G, L289P; S2P, M71A, D264G, F312L; S2P, M71A, D264G, T348A;S2P, M71A, D264G, L380F; S2P, M71A, D264G, T394M; S2P, M71A, D264G,F396Y; S2P, M71A, D264G, R400C; S2P, M71A, D264G, T413A; S2P, M71A,D264G, E423D; S2P, M71A, D264G, L453P; S2P, M71A, D264G, F458S; S2P,M71A, D264G, E461G; S2P, M71A, D264G, F465C; S2P, M71A, D264G, N467T;S2P, M71A, D264G, N470S; S2P, M71A, D264G, A476T; S2P, M71A, D264G,Q494R; S2P, M71A, D264G, T496R; S2P, M71A, D264G, G504S; S2P, M71C,D264G, T283A; S2P, M71C, D264G, L289P; S2P, M71C, D264G, F312L; S2P,M71C, D264G, T348A; S2P, M71C, D264G, L380F; S2P, M71C, D264G, T394M;S2P, M71C, D264G, F396Y; S2P, M71C, D264G, R400C; S2P, M71C, D264G,T413A; S2P, M71C, D264G, E423D; S2P, M71C, D264G, L453P; S2P, M71C,D264G, F458S; S2P, M71C, D264G, E461G; S2P, M71C, D264G, F465C; S2P,M71C, D264G, N467T; S2P, M71C, D264G, N470S; S2P, M71C, D264G, A476T;S2P, M71C, D264G, Q494R; S2P, M71C, D264G, T496R; S2P, M71C, D264G,G504S; S2P, M71V, T283A, L289P; S2P, M71V, T283A, F312L; S2P, M71V,T283A, T348A; S2P, M71V, T283A, L380F; S2P, M71V, T283A, T394M; S2P,M71V, T283A, F396Y; S2P, M71V, T283A, R400C; S2P, M71V, T283A, T413A;S2P, M71V, T283A, E423D; S2P, M71V, T283A, L453P; S2P, M71V, T283A,F458S; S2P, M71V, T283A, E461G; S2P, M71V, T283A, F465C; S2P, M71V,T283A, N467T; S2P, M71V, T283A, N470S; S2P, M71V, T283A, A476T; S2P,M71V, T283A, Q494R; S2P, M71V, T283A, T496R; S2P, M71V, T283A, G504S;S2P, M71T, T283A, L289P; S2P, M71T, T283A, F312L; S2P, M71T, T283A,T348A; S2P, M71T, T283A, L380F; S2P, M71T, T283A, T394M; S2P, M71T,T283A, F396Y; S2P, M71T, T283A, R400C; S2P, M71T, T283A, T413A; S2P,M71T, T283A, E423D; S2P, M71T, T283A, L453P; S2P, M71T, T283A, F458S;S2P, M71T, T283A, E461G; S2P, M71T, T283A, F465C; S2P, M71T, T283A,N467T; S2P, M71T, T283A, N470S; S2P, M71T, T283A, A476T; S2P, M71T,T283A, Q494R; S2P, M71T, T283A, T496R; S2P, M71T, T283A, G504S; S2P,M71A, T283A, L289P; S2P, M71A, T283A, F312L; S2P, M71A, T283A, T348A;S2P, M71A, T283A, L380F; S2P, M71A, T283A, T394M; S2P, M71A, T283A,F396Y; S2P, M71A, T283A, R400C; S2P, M71A, T283A, T413A; S2P, M71A,T283A, E423D; S2P, M71A, T283A, L453P; S2P, M71A, T283A, F458S; S2P,M71A, T283A, E461G; S2P, M71A, T283A, F465C; S2P, M71A, T283A, N467T;S2P, M71A, T283A, N470S; S2P, M71A, T283A, A476T; S2P, M71A, T283A,Q494R; S2P, M71A, T283A, T496R; S2P, M71A, T283A, G504S; S2P, M71C,T283A, L289P; S2P, M71C, T283A, F312L; S2P, M71C, T283A, T348A; S2P,M71C, T283A, L380F; S2P, M71C, T283A, T394M; S2P, M71C, T283A, F396Y;S2P, M71C, T283A, R400C; S2P, M71C, T283A, T413A; S2P, M71C, T283A,E423D; S2P, M71C, T283A, L453P; S2P, M71C, T283A, F458S; S2P, M71C,T283A, E461G; S2P, M71C, T283A, F465C; S2P, M71C, T283A, N467T; S2P,M71C, T283A, N470S; S2P, M71C, T283A, A476T; S2P, M71C, T283A, Q494R;S2P, M71C, T283A, T496R; S2P, M71C, T283A, G504S; S2P, M71V, L289P,F312L; S2P, M71V, L289P, T348A; S2P, M71V, L289P, L380F; S2P, M71V,L289P, T394M; S2P, M71V, L289P, F396Y; S2P, M71V, L289P, R400C; S2P,M71V, L289P, T413A; S2P, M71V, L289P, E423D; S2P, M71V, L289P, L453P;S2P, M71V, L289P, F458S; S2P, M71V, L289P, E461G; S2P, M71V, L289P,F465C; S2P, M71V, L289P, N467T; S2P, M71V, L289P, N470S; S2P, M71V,L289P, A476T; S2P, M71V, L289P, Q494R; S2P, M71V, L289P, T496R; S2P,M71V, L289P, G504S; S2P, M71T, L289P, F312L; S2P, M71T, L289P, T348A;S2P, M71T, L289P, L380F; S2P, M71T, L289P, T394M; S2P, M71T, L289P,F396Y; S2P, M71T, L289P, R400C; S2P, M71T, L289P, T413A; S2P, M71T,L289P, E423D; S2P, M71T, L289P, L453P; S2P, M71T, L289P, F458S; S2P,M71T, L289P, E461G; S2P, M71T, L289P, F465C; S2P, M71T, L289P, N467T;S2P, M71T, L289P, N470S; S2P, M71T, L289P, A476T; S2P, M71T, L289P,Q494R; S2P, M71T, L289P, T496R; S2P, M71T, L289P, G504S; S2P, M71A,L289P, F312L; S2P, M71A, L289P, T348A; S2P, M71A, L289P, L380F; S2P,M71A, L289P, T394M; S2P, M71A, L289P, F396Y; S2P, M71A, L289P, R400C;S2P, M71A, L289P, T413A; S2P, M71A, L289P, E423D; S2P, M71A, L289P,L453P; S2P, M71A, L289P, F458S; S2P, M71A, L289P, E461G; S2P, M71A,L289P, F465C; S2P, M71A, L289P, N467T; S2P, M71A, L289P, N470S; S2P,M71A, L289P, A476T; S2P, M71A, L289P, Q494R; S2P, M71A, L289P, T496R;S2P, M71A, L289P, G504S; S2P, M71C, L289P, F312L; S2P, M71C, L289P,T348A; S2P, M71C, L289P, L380F; S2P, M71C, L289P, T394M; S2P, M71C,L289P, F396Y; S2P, M71C, L289P, R400C; S2P, M71C, L289P, T413A; S2P,M71C, L289P, E423D; S2P, M71C, L289P, L453P; S2P, M71C, L289P, F458S;S2P, M71C, L289P, E461G; S2P, M71C, L289P, F465C; S2P, M71C, L289P,N467T; S2P, M71C, L289P, N470S; S2P, M71C, L289P, A476T; S2P, M71C,L289P, Q494R; S2P, M71C, L289P, T496R; S2P, M71C, L289P, G504S; S2P,M71V, F312L, T348A; S2P, M71V, F312L, L380F; S2P, M71V, F312L, T394M;S2P, M71V, F312L, F396Y; S2P, M71V, F312L, R400C; S2P, M71V, F312L,T413A; S2P, M71V, F312L, E423D; S2P, M71V, F312L, L453P; S2P, M71V,F312L, F458S; S2P, M71V, F312L, E461G; S2P, M71V, F312L, F465C; S2P,M71V, F312L, N467T; S2P, M71V, F312L, N470S; S2P, M71V, F312L, A476T;S2P, M71V, F312L, Q494R; S2P, M71V, F312L, T496R; S2P, M71V, F312L,G504S; S2P, M71T, F312L, T348A; S2P, M71T, F312L, L380F; S2P, M71T,F312L, T394M; S2P, M71T, F312L, F396Y; S2P, M71T, F312L, R400C; S2P,M71T, F312L, T413A; S2P, M71T, F312L, E423D; S2P, M71T, F312L, L453P;S2P, M71T, F312L, F458S; S2P, M71T, F312L, E461G; S2P, M71T, F312L,F465C; S2P, M71T, F312L, N467T; S2P, M71T, F312L, N470S; S2P, M71T,F312L, A476T; S2P, M71T, F312L, Q494R; S2P, M71T, F312L, T496R; S2P,M71T, F312L, G504S; S2P, M71A, F312L, T348A; S2P, M71A, F312L, L380F;S2P, M71A, F312L, T394M; S2P, M71A, F312L, F396Y; S2P, M71A, F312L,R400C; S2P, M71A, F312L, T413A; S2P, M71A, F312L, E423D; S2P, M71A,F312L, L453P; S2P, M71A, F312L, F458S; S2P, M71A, F312L, E461G; S2P,M71A, F312L, F465C; S2P, M71A, F312L, N467T; S2P, M71A, F312L, N470S;S2P, M71A, F312L, A476T; S2P, M71A, F312L, Q494R; S2P, M71A, F312L,T496R; S2P, M71A, F312L, G504S; S2P, M71C, F312L, T348A; S2P, M71C,F312L, L380F; S2P, M71C, F312L, T394M; S2P, M71C, F312L, F396Y; S2P,M71C, F312L, R400C; S2P, M71C, F312L, T413A; S2P, M71C, F312L, E423D;S2P, M71C, F312L, L453P; S2P, M71C, F312L, F458S; S2P, M71C, F312L,E461G; S2P, M71C, F312L, F465C; S2P, M71C, F312L, N467T; S2P, M71C,F312L, N470S; S2P, M71C, F312L, A476T; S2P, M71C, F312L, Q494R; S2P,M71C, F312L, T496R; S2P, M71C, F312L, G504S; S2P, M71V, T348A, L380F;S2P, M71V, T348A, T394M; S2P, M71V, T348A, F396Y; S2P, M71V, T348A,R400C; S2P, M71V, T348A, T413A; S2P, M71V, T348A, E423D; S2P, M71V,T348A, L453P; S2P, M71V, T348A, F458S; S2P, M71V, T348A, E461G; S2P,M71V, T348A, F465C; S2P, M71V, T348A, N467T; S2P, M71V, T348A, N470S;S2P, M71V, T348A, A476T; S2P, M71V, T348A, Q494R; S2P, M71V, T348A,T496R; S2P, M71V, T348A, G504S; S2P, M71T, T348A, L380F; S2P, M71T,T348A, T394M; S2P, M71T, T348A, F396Y; S2P, M71T, T348A, R400C; S2P,M71T, T348A, T413A; S2P, M71T, T348A, E423D; S2P, M71T, T348A, L453P;S2P, M71T, T348A, F458S; S2P, M71T, T348A, E461G; S2P, M71T, T348A,F465C; S2P, M71T, T348A, N467T; S2P, M71T, T348A, N470S; S2P, M71T,T348A, A476T; S2P, M71T, T348A, Q494R; S2P, M71T, T348A, T496R; S2P,M71T, T348A, G504S; S2P, M71A, T348A, L380F; S2P, M71A, T348A, T394M;S2P, M71A, T348A, F396Y; S2P, M71A, T348A, R400C; S2P, M71A, T348A,T413A; S2P, M71A, T348A, E423D; S2P, M71A, T348A, L453P; S2P, M71A,T348A, F458S; S2P, M71A, T348A, E461G; S2P, M71A, T348A, F465C; S2P,M71A, T348A, N467T; S2P, M71A, T348A, N470S; S2P, M71A, T348A, A476T;S2P, M71A, T348A, Q494R; S2P, M71A, T348A, T496R; S2P, M71A, T348A,G504S; S2P, M71C, T348A, L380F; S2P, M71C, T348A, T394M; S2P, M71C,T348A, F396Y; S2P, M71C, T348A, R400C; S2P, M71C, T348A, T413A; S2P,M71C, T348A, E423D; S2P, M71C, T348A, L453P; S2P, M71C, T348A, F458S;S2P, M71C, T348A, E461G; S2P, M71C, T348A, F465C; S2P, M71C, T348A,N467T; S2P, M71C, T348A, N470S; S2P, M71C, T348A, A476T; S2P, M71C,T348A, Q494R; S2P, M71C, T348A, T496R; S2P, M71C, T348A, G504S; S2P,M71V, L380F, T394M; S2P, M71V, L380F, F396Y; S2P, M71V, L380F, R400C;S2P, M71V, L380F, T413A; S2P, M71V, L380F, E423D; S2P, M71V, L380F,L453P; S2P, M71V, L380F, F458S; S2P, M71V, L380F, E461G; S2P, M71V,L380F, F465C; S2P, M71V, L380F, N467T; S2P, M71V, L380F, N470S; S2P,M71V, L380F, A476T; S2P, M71V, L380F, Q494R; S2P, M71V, L380F, T496R;S2P, M71V, L380F, G504S; S2P, M71T, L380F, T394M; S2P, M71T, L380F,F396Y; S2P, M71T, L380F, R400C; S2P, M71T, L380F, T413A; S2P, M71T,L380F, E423D; S2P, M71T, L380F, L453P; S2P, M71T, L380F, F458S; S2P,M71T, L380F, E461G; S2P, M71T, L380F, F465C; S2P, M71T, L380F, N467T;S2P, M71T, L380F, N470S; S2P, M71T, L380F, A476T; S2P, M71T, L380F,Q494R; S2P, M71T, L380F, T496R; S2P, M71T, L380F, G504S; S2P, M71A,L380F, T394M; S2P, M71A, L380F, F396Y; S2P, M71A, L380F, R400C; S2P,M71A, L380F, T413A; S2P, M71A, L380F, E423D; S2P, M71A, L380F, L453P;S2P, M71A, L380F, F458S; S2P, M71A, L380F, E461G; S2P, M71A, L380F,F465C; S2P, M71A, L380F, N467T; S2P, M71A, L380F, N470S; S2P, M71A,L380F, A476T; S2P, M71A, L380F, Q494R; S2P, M71A, L380F, T496R; S2P,M71A, L380F, G504S; S2P, M71C, L380F, T394M; S2P, M71C, L380F, F396Y;S2P, M71C, L380F, R400C; S2P, M71C, L380F, T413A; S2P, M71C, L380F,E423D; S2P, M71C, L380F, L453P; S2P, M71C, L380F, F458S; S2P, M71C,L380F, E461G; S2P, M71C, L380F, F465C; S2P, M71C, L380F, N467T; S2P,M71C, L380F, N470S; S2P, M71C, L380F, A476T; S2P, M71C, L380F, Q494R;S2P, M71C, L380F, T496R; S2P, M71C, L380F, G504S; S2P, M71V, T394M,F396Y; S2P, M71V, T394M, R400C; S2P, M71V, T394M, T413A; S2P, M71V,T394M, E423D; S2P, M71V, T394M, L453P; S2P, M71V, T394M, F458S; S2P,M71V, T394M, E461G; S2P, M71V, T394M, F465C; S2P, M71V, T394M, N467T;S2P, M71V, T394M, N470S; S2P, M71V, T394M, A476T; S2P, M71V, T394M,Q494R; S2P, M71V, T394M, T496R; S2P, M71V, T394M, G504S; S2P, M71T,T394M, F396Y; S2P, M71T, T394M, R400C; S2P, M71T, T394M, T413A; S2P,M71T, T394M, E423D; S2P, M71T, T394M, L453P; S2P, M71T, T394M, F458S;S2P, M71T, T394M, E461G; S2P, M71T, T394M, F465C; S2P, M71T, T394M,N467T; S2P, M71T, T394M, N470S; S2P, M71T, T394M, A476T; S2P, M71T,T394M, Q494R; S2P, M71T, T394M, T496R; S2P, M71T, T394M, G504S; S2P,M71A, T394M, F396Y; S2P, M71A, T394M, R400C; S2P, M71A, T394M, T413A;S2P, M71A, T394M, E423D; S2P, M71A, T394M, L453P; S2P, M71A, T394M,F458S; S2P, M71A, T394M, E461G; S2P, M71A, T394M, F465C; S2P, M71A,T394M, N467T; S2P, M71A, T394M, N470S; S2P, M71A, T394M, A476T; S2P,M71A, T394M, Q494R; S2P, M71A, T394M, T496R; S2P, M71A, T394M, G504S;S2P, M71C, T394M, F396Y; S2P, M71C, T394M, R400C; S2P, M71C, T394M,T413A; S2P, M71C, T394M, E423D; S2P, M71C, T394M, L453P; S2P, M71C,T394M, F458S; S2P, M71C, T394M, E461G; S2P, M71C, T394M, F465C; S2P,M71C, T394M, N467T; S2P, M71C, T394M, N470S; S2P, M71C, T394M, A476T;S2P, M71C, T394M, Q494R; S2P, M71C, T394M, T496R; S2P, M71C, T394M,G504S; S2P, M71V, F396Y, R400C; S2P, M71V, F396Y, T413A; S2P, M71V,F396Y, E423D; S2P, M71V, F396Y, L453P; S2P, M71V, F396Y, F458S; S2P,M71V, F396Y, E461G; S2P, M71V, F396Y, F465C; S2P, M71V, F396Y, N467T;S2P, M71V, F396Y, N470S; S2P, M71V, F396Y, A476T; S2P, M71V, F396Y,Q494R; S2P, M71V, F396Y, T496R; S2P, M71V, F396Y, G504S; S2P, M71T,F396Y, R400C; S2P, M71T, F396Y, T413A; S2P, M71T, F396Y, E423D; S2P,M71T, F396Y, L453P; S2P, M71T, F396Y, F458S; S2P, M71T, F396Y, E461G;S2P, M71T, F396Y, F465C; S2P, M71T, F396Y, N467T; S2P, M71T, F396Y,N470S; S2P, M71T, F396Y, A476T; S2P, M71T, F396Y, Q494R; S2P, M71T,F396Y, T496R; S2P, M71T, F396Y, G504S; S2P, M71A, F396Y, R400C; S2P,M71A, F396Y, T413A; S2P, M71A, F396Y, E423D; S2P, M71A, F396Y, L453P;S2P, M71A, F396Y, F458S; S2P, M71A, F396Y, E461G; S2P, M71A, F396Y,F465C; S2P, M71A, F396Y, N467T; S2P, M71A, F396Y, N470S; S2P, M71A,F396Y, A476T; S2P, M71A, F396Y, Q494R; S2P, M71A, F396Y, T496R; S2P,M71A, F396Y, G504S; S2P, M71C, F396Y, R400C; S2P, M71C, F396Y, T413A;S2P, M71C, F396Y, E423D; S2P, M71C, F396Y, L453P; S2P, M71C, F396Y,F458S; S2P, M71C, F396Y, E461G; S2P, M71C, F396Y, F465C; S2P, M71C,F396Y, N467T; S2P, M71C, F396Y, N470S; S2P, M71C, F396Y, A476T; S2P,M71C, F396Y, Q494R; S2P, M71C, F396Y, T496R; S2P, M71C, F396Y, G504S;S2P, M71V, R400C, T413A; S2P, M71V, R400C, E423D; S2P, M71V, R400C,L453P; S2P, M71V, R400C, F458S; S2P, M71V, R400C, E461G; S2P, M71V,R400C, F465C; S2P, M71V, R400C, N467T; S2P, M71V, R400C, N470S; S2P,M71V, R400C, A476T; S2P, M71V, R400C, Q494R; S2P, M71V, R400C, T496R;S2P, M71V, R400C, G504S; S2P, M71T, R400C, T413A; S2P, M71T, R400C,E423D; S2P, M71T, R400C, L453P; S2P, M71T, R400C, F458S; S2P, M71T,R400C, E461G; S2P, M71T, R400C, F465C; S2P, M71T, R400C, N467T; S2P,M71T, R400C, N470S; S2P, M71T, R400C, A476T; S2P, M71T, R400C, Q494R;S2P, M71T, R400C, T496R; S2P, M71T, R400C, G504S; S2P, M71A, R400C,T413A; S2P, M71A, R400C, E423D; S2P, M71A, R400C, L453P; S2P, M71A,R400C, F458S; S2P, M71A, R400C, E461G; S2P, M71A, R400C, F465C; S2P,M71A, R400C, N467T; S2P, M71A, R400C, N470S; S2P, M71A, R400C, A476T;S2P, M71A, R400C, Q494R; S2P, M71A, R400C, T496R; S2P, M71A, R400C,G504S; S2P, M71C, R400C, T413A; S2P, M71C, R400C, E423D; S2P, M71C,R400C, L453P; S2P, M71C, R400C, F458S; S2P, M71C, R400C, E461G; S2P,M71C, R400C, F465C; S2P, M71C, R400C, N467T; S2P, M71C, R400C, N470S;S2P, M71C, R400C, A476T; S2P, M71C, R400C, Q494R; S2P, M71C, R400C,T496R; S2P, M71C, R400C, G504S; S2P, M71V, T413A, E423D; S2P, M71V,T413A, L453P; S2P, M71V, T413A, F458S; S2P, M71V, T413A, E461G; S2P,M71V, T413A, F465C; S2P, M71V, T413A, N467T; S2P, M71V, T413A, N470S;S2P, M71V, T413A, A476T; S2P, M71V, T413A, Q494R; S2P, M71V, T413A,T496R; S2P, M71V, T413A, G504S; S2P, M71T, T413A, E423D; S2P, M71T,T413A, L453P; S2P, M71T, T413A, F458S; S2P, M71T, T413A, E461G; S2P,M71T, T413A, F465C; S2P, M71T, T413A, N467T; S2P, M71T, T413A, N470S;S2P, M71T, T413A, A476T; S2P, M71T, T413A, Q494R; S2P, M71T, T413A,T496R; S2P, M71T, T413A, G504S; S2P, M71A, T413A, E423D; S2P, M71A,T413A, L453P; S2P, M71A, T413A, F458S; S2P, M71A, T413A, E461G; S2P,M71A, T413A, F465C; S2P, M71A, T413A, N467T; S2P, M71A, T413A, N470S;S2P, M71A, T413A, A476T; S2P, M71A, T413A, Q494R; S2P, M71A, T413A,T496R; S2P, M71A, T413A, G504S; S2P, M71C, T413A, E423D; S2P, M71C,T413A, L453P; S2P, M71C, T413A, F458S; S2P, M71C, T413A, E461G; S2P,M71C, T413A, F465C; S2P, M71C, T413A, N467T; S2P, M71C, T413A, N470S;S2P, M71C, T413A, A476T; S2P, M71C, T413A, Q494R; S2P, M71C, T413A,T496R; S2P, M71C, T413A, G504S; S2P, M71V, E423D, L453P; S2P, M71V,E423D, F458S; S2P, M71V, E423D, E461G; S2P, M71V, E423D, F465C; S2P,M71V, E423D, N467T; S2P, M71V, E423D, N470S; S2P, M71V, E423D, A476T;S2P, M71V, E423D, Q494R; S2P, M71V, E423D, T496R; S2P, M71V, E423D,G504S; S2P, M71T, E423D, L453P; S2P, M71T, E423D, F458S; S2P, M71T,E423D, E461G; S2P, M71T, E423D, F465C; S2P, M71T, E423D, N467T; S2P,M71T, E423D, N470S; S2P, M71T, E423D, A476T; S2P, M71T, E423D, Q494R;S2P, M71T, E423D, T496R; S2P, M71T, E423D, G504S; S2P, M71A, E423D,L453P; S2P, M71A, E423D, F458S; S2P, M71A, E423D, E461G; S2P, M71A,E423D, F465C; S2P, M71A, E423D, N467T; S2P, M71A, E423D, N470S; S2P,M71A, E423D, A476T; S2P, M71A, E423D, Q494R; S2P, M71A, E423D, T496R;S2P, M71A, E423D, G504S; S2P, M71C, E423D, L453P; S2P, M71C, E423D,F458S; S2P, M71C, E423D, E461G; S2P, M71C, E423D, F465C; S2P, M71C,E423D, N467T; S2P, M71C, E423D, N470S; S2P, M71C, E423D, A476T; S2P,M71C, E423D, Q494R; S2P, M71C, E423D, T496R; S2P, M71C, E423D, G504S;S2P, M71V, L453P, F458S; S2P, M71V, L453P, E461G; S2P, M71V, L453P,F465C; S2P, M71V, L453P, N467T; S2P, M71V, L453P, N470S; S2P, M71V,L453P, A476T; S2P, M71V, L453P, Q494R; S2P, M71V, L453P, T496R; S2P,M71V, L453P, G504S; S2P, M71T, L453P, F458S; S2P, M71T, L453P, E461G;S2P, M71T, L453P, F465C; S2P, M71T, L453P, N467T; S2P, M71T, L453P,N470S; S2P, M71T, L453P, A476T, S2P, M71T, L453P, Q494R; S2P, M71T,L453P, T496R; S2P, M71T, L453P, G504S; S2P, M71A, L453P, F458S; S2P,M71A, L453P, E461G; S2P, M71A, L453P, F465C; S2P, M71A, L453P, N467T;S2P, M71A, L453P, N470S; S2P, M71A, L453P, A476T; S2P, M71A, L453P,Q494R; S2P, M71A, L453P, T496R; S2P, M71A, L453P, G504S; S2P, M71C,L453P, F458S; S2P, M71C, L453P, E461G; S2P, M71C, L453P, F465C; S2P,M71C, L453P, N467T; S2P, M71C, L453P, N470S; S2P, M71C, L453P, A476T;S2P, M71C, L453P, Q494R; S2P, M71C, L453P, T496R; S2P, M71C, L453P,G504S; S2P, M71V, F458S, E461G; S2P, M71V, F458S, F465C; S2P, M71V,F458S, N467T; S2P, M71V, F458S, N470S; S2P, M71V, F458S, A476T; S2P,M71V, F458S, Q494R; S2P, M71V, F458S, T496R; S2P, M71V, F458S, G504S;S2P, M71T, F458S, E461G; S2P, M71T, F458S, F465C; S2P, M71T, F458S,N467T; S2P, M71T, F458S, N470S; S2P, M71T, F458S, A476T; S2P, M71T,F458S, Q494R; S2P, M71T, F458S, T496R; S2P, M71T, F458S, G504S; S2P,M71A, F458S, E461G; S2P, M71A, F458S, F465C; S2P, M71A, F458S, N467T;S2P, M71A, F458S, N470S; S2P, M71A, F458S, A476T; S2P, M71A, F458S,Q494R; S2P, M71A, F458S, T496R; S2P, M71A, F458S, G504S; S2P, M71C,F458S, E461G; S2P, M71C, F458S, F465C; S2P, M71C, F458S, N467T; S2P,M71C, F458S, N470S; S2P, M71C, F458S, A476T; S2P, M71C, F458S, Q494R;S2P, M71C, F458S, T496R; S2P, M71C, F458S, G504S; S2P, M71V, E461G,F465C; S2P, M71V, E461G, N467T; S2P, M71V, E461G, N470S; S2P, M71V,E461G, A476T; S2P, M71V, E461G, Q494R; S2P, M71V, E461G, T496R; S2P,M71V, E461G, G504S; S2P, M71T, E461G, F465C; S2P, M71T, E461G, N467T;S2P, M71T, E461G, N470S; S2P, M71T, E461G, A476T; S2P, M71T, E461G,Q494R; S2P, M71T, E461G, T496R; S2P, M71T, E461G, G504S; S2P, M71A,E461G, F465C; S2P, M71A, E461G, N467T; S2P, M71A, E461G, N470S; S2P,M71A, E461G, A476T; S2P, M71A, E461G, Q494R; S2P, M71A, E461G, T496R;S2P, M71A, E461G, G504S; S2P, M71C, E461G, F465C; S2P, M71C, E461G,N467T; S2P, M71C, E461G, N470S; S2P, M71C, E461G, A476T; S2P, M71C,E461G, Q494R; S2P, M71C, E461G, T496R; S2P, M71C, E461G, G504S; S2P,M71V, F465C, N467T; S2P, M71V, F465C, N470S; S2P, M71V, F465C, A476T;S2P, M71V, F465C, Q494R; S2P, M71V, F465C, T496R; S2P, M71V, F465C,G504S; S2P, M71T, F465C, N467T; S2P, M71T, F465C, N470S; S2P, M71T,F465C, A476T; S2P, M71T, F465C, Q494R; S2P, M71T, F465C, T496R; S2P,M71T, F465C, G504S; S2P, M71A, F465C, N467T; S2P, M71A, F465C, N470S;S2P, M71A, F465C, A476T; S2P, M71A, F465C, Q494R; S2P, M71A, F465C,T496R; S2P, M71A, F465C, G504S; S2P, M71C, F465C, N467T; S2P, M71C,F465C, N470S; S2P, M71C, F465C, A476T; S2P, M71C, F465C, Q494R; S2P,M71C, F465C, T496R; S2P, M71C, F465C, G504S; S2P, M71V, N467T, N470S;S2P, M71V, N467T, A476T; S2P, M71V, N467T, Q494R; S2P, M71V, N467T,T496R; S2P, M71V, N467T, G504S; S2P, M71T, N467T, N470S; S2P, M71T,N467T, A476T; S2P, M71T, N467T, Q494R; S2P, M71T, N467T, T496R; S2P,M71T, N467T, G504S; S2P, M71A, N467T, N470S; S2P, M71A, N467T, A476T;S2P, M71A, N467T, Q494R; S2P, M71A, N467T, T496R; S2P, M71A, N467T,G504S; S2P, M71C, N467T, N470S; S2P, M71C, N467T, A476T; S2P, M71C,N467T, Q494R; S2P, M71C, N467T, T496R; S2P, M71C, N467T, G504S; S2P,M71V, N470S, A476T; S2P, M71V, N470S, Q494R; S2P, M71V, N470S, T496R;S2P, M71V, N470S, G504S; S2P, M71T, N470S, A476T; S2P, M71T, N470S,Q494R; S2P, M71T, N470S, T496R; S2P, M71T, N470S, G504S; S2P, M71A,N470S, A476T; S2P, M71A, N470S, Q494R; S2P, M71A, N470S, T496R; S2P,M71A, N470S, G504S; S2P, M71C, N470S, A476T; S2P, M71C, N470S, Q494R;S2P, M71C, N470S, T496R; S2P, M71C, N470S, G504S; S2P, M71V, A476T,Q494R; S2P, M71V, A476T, T496R; S2P, M71V, A476T, G504S; S2P, M71T,A476T, Q494R; S2P, M71T, A476T, T496R; S2P, M71T, A476T, G504S; S2P,M71A, A476T, Q494R; S2P, M71A, A476T, T496R; S2P, M71A, A476T, G504S;S2P, M71C, A476T, Q494R; S2P, M71C, A476T, T496R; S2P, M71C, A476T,G504S; S2P, M71V, Q494R, T496R; S2P, M71V, Q494R, G504S; S2P, M71T,Q494R, T496R; S2P, M71T, Q494R, G504S; S2P, M71A, Q494R, T496R; S2P,M71A, Q494R, G504S; S2P, M71C, Q494R, T496R; S2P, M71C, Q494R, G504S;S2P, M71V, T496R, G504S; S2P, M71T, T496R, G504S; S2P, M71A, T496R,G504S; S2P, M71C, T496R, G504S; S2P, M71T, N75K, E96V; S2P, M71A, N75K,E96V; S2P, M71C, N75K, E96V; S2P, N75K, E96V, D101G; S2P, N75K, E96V,G112S; S2P, N75K, E96V, L114P; S2P, N75K, E96V, S130L; S2P, N75K, E96V,K145M; S2P, N75K, E96V, N166S; S2P, N75K, E96V, F171I; S2P, N75K, E96V,D203A; S2P, N75K, E96V, D203G; S2P, N75K, E96V, T213A; S2P, N75K, E96V,V225I; S2P, N75K, E96V, K237E; S2P, N75K, E96V, V256I; S2P, N75K, E96V,T258A; S2P, N75K, E96V, D264G; S2P, N75K, E96V, T283A; S2P, N75K, E96V,L289P; S2P, N75K, E96V, F312L; S2P, N75K, E96V, T348A; S2P, N75K, E96V,L380F; S2P, N75K, E96V, T394M; S2P, N75K, E96V, F396Y; S2P, N75K, E96V,R400C; S2P, N75K, E96V, T413A; S2P, N75K, E96V, E423D; S2P, N75K, E96V,L453P; S2P, N75K, E96V, F458S; S2P, N75K, E96V, E461G; S2P, N75K, E96V,F465C; S2P, N75K, E96V, N467T; S2P, N75K, E96V, N470S; S2P, N75K, E96V,A476T; S2P, N75K, E96V, Q494R; S2P, N75K, E96V, T496R; S2P, N75K, E96V,G504S; M71V, N75K, E96V, D101G; M71V, N75K, E96V, G112S; M71V, N75K,E96V, L114P; M71V, N75K, E96V, S130L; M71V, N75K, E96V, K145M; M71V,N75K, E96V, N166S; M71V, N75K, E96V, F171I; M71V, N75K, E96V, D203A;M71V, N75K, E96V, D203G; M71V, N75K, E96V, T213A; M71V, N75K, E96V,V225I; M71V, N75K, E96V, K237E; M71V, N75K, E96V, V256I; M71V, N75K,E96V, T258A; M71V, N75K, E96V, D264G; M71V, N75K, E96V, T283A; M71V,N75K, E96V, L289P; M71V, N75K, E96V, F312L; M71V, N75K, E96V, T348A;M71V, N75K, E96V, L380F; M71V, N75K, E96V, T394M; M71V, N75K, E96V,F396Y; M71V, N75K, E96V, R400C; M71V, N75K, E96V, T413A; M71V, N75K,E96V, E423D; M71V, N75K, E96V, L453P; M71V, N75K, E96V, F458S; M71V,N75K, E96V, E461G; M71V, N75K, E96V, F465C; M71V, N75K, E96V, N467T;M71V, N75K, E96V, N470S; M71V, N75K, E96V, A476T; M71V, N75K, E96V,Q494R; M71V, N75K, E96V, T496R; M71V, N75K, E96V, G504S; M71T, N75K,E96V, D101G; M71T, N75K, E96V, G112S; M71T, N75K, E96V, L114P; M71T,N75K, E96V, S130L; M71T, N75K, E96V, K145M; M71T, N75K, E96V, N166S;M71T, N75K, E96V, F171I; M71T, N75K, E96V, D203A; M71T, N75K, E96V,D203G; M71T, N75K, E96V, T213A; M71T, N75K, E96V, V225I; M71T, N75K,E96V, K237E; M71T, N75K, E96V, V256I; M71T, N75K, E96V, T258A; M71T,N75K, E96V, D264G; M71T, N75K, E96V, T283A; M71T, N75K, E96V, L289P;M71T, N75K, E96V, F312L; M71T, N75K, E96V, T348A; M71T, N75K, E96V,L380F; M71T, N75K, E96V, T394M; M71T, N75K, E96V, F396Y; M71T, N75K,E96V, R400C; M71T, N75K, E96V, T413A; M71T, N75K, E96V, E423D; M71T,N75K, E96V, L453P; M71T, N75K, E96V, F458S; M71T, N75K, E96V, E461G;M71T, N75K, E96V, F465C; M71T, N75K, E96V, N467T; M71T, N75K, E96V,N470S; M71T, N75K, E96V, A476T; M71T, N75K, E96V, Q494R; M71T, N75K,E96V, T496R; M71T, N75K, E96V, G504S; M71A, N75K, E96V, D101G; M71A,N75K, E96V, G112S; M71A, N75K, E96V, L114P; M71A, N75K, E96V, S130L;M71A, N75K, E96V, K145M; M71A, N75K, E96V, N166S; M71A, N75K, E96V,F171I; M71A, N75K, E96V, D203A; M71A, N75K, E96V, D203G; M71A, N75K,E96V, T213A; M71A, N75K, E96V, V225I; M71A, N75K, E96V, K237E; M71A,N75K, E96V, V256I; M71A, N75K, E96V, T258A; M71A, N75K, E96V, D264G;M71A, N75K, E96V, T283A; M71A, N75K, E96V, L289P; M71A, N75K, E96V,F312L; M71A, N75K, E96V, T348A; M71A, N75K, E96V, L380F; M71A, N75K,E96V, T394M; M71A, N75K, E96V, F396Y; M71A, N75K, E96V, R400C; M71A,N75K, E96V, T413A; M71A, N75K, E96V, E423D; M71A, N75K, E96V, L453P;M71A, N75K, E96V, F458S; M71A, N75K, E96V, E461G; M71A, N75K, E96V,F465C; M71A, N75K, E96V, N467T; M71A, N75K, E96V, N470S; M71A, N75K,E96V, A476T; M71A, N75K, E96V, Q494R; M71A, N75K, E96V, T496R; M71A,N75K, E96V, G504S; M71C, N75K, E96V, D101G; M71C, N75K, E96V, G112S;M71C, N75K, E96V, L114P; M71C, N75K, E96V, S130L; M71C, N75K, E96V,K145M; M71C, N75K, E96V, N166S; M71C, N75K, E96V, F171I; M71C, N75K,E96V, D203A; M71C, N75K, E96V, D203G; M71C, N75K, E96V, T213A; M71C,N75K, E96V, V225I; M71C, N75K, E96V, K237E; M71C, N75K, E96V, V256I;M71C, N75K, E96V, T258A; M71C, N75K, E96V, D264G; M71C, N75K, E96V,T283A; M71C, N75K, E96V, L289P; M71C, N75K, E96V, F312L; M71C, N75K,E96V, T348A; M71C, N75K, E96V, L380F; M71C, N75K, E96V, T394M; M71C,N75K, E96V, F396Y; M71C, N75K, E96V, R400C; M71C, N75K, E96V, T413A;M71C, N75K, E96V, E423D; M71C, N75K, E96V, L453P; M71C, N75K, E96V,F458S; M71C, N75K, E96V, E461G; M71C, N75K, E96V, F465C; M71C, N75K,E96V, N467T; M71C, N75K, E96V, N470S; M71C, N75K, E96V, A476T; M71C,N75K, E96V, Q494R; M71C, N75K, E96V, T496R; M71C, N75K, E96V, G504S;N75K, E96V, D101G, G112S; N75K, E96V, D101G, L114P; N75K, E96V, D101G,S130L; N75K, E96V, D101G, K145M; N75K, E96V, D101G, N166S; N75K, E96V,D101G, F171I; N75K, E96V, D101G, D203A; N75K, E96V, D101G, D203G; N75K,E96V, D101G, T213A; N75K, E96V, D101G, V225I; N75K, E96V, D101G, K237E;N75K, E96V, D101G, V256I; N75K, E96V, D101G, T258A; N75K, E96V, D101G,D264G; N75K, E96V, D101G, T283A; N75K, E96V, D101G, L289P; N75K, E96V,D101G, F312L; N75K, E96V, D101G, T348A; N75K, E96V, D101G, L380F; N75K,E96V, D101G, T394M; N75K, E96V, D101G, F396Y; N75K, E96V, D101G, R400C;N75K, E96V, D101G, T413A; N75K, E96V, D101G, E423D; N75K, E96V, D101G,L453P; N75K, E96V, D101G, F458S; N75K, E96V, D101G, E461G; N75K, E96V,D101G, F465C; N75K, E96V, D101G, N467T; N75K, E96V, D101G, N470S; N75K,E96V, D101G, A476T; N75K, E96V, D101G, Q494R; N75K, E96V, D101G, T496R;N75K, E96V, D101G, G504S; E96V, D101G, G112S, L114P; E96V, D101G, G112S,S130L; E96V, D101G, G112S, K145M; E96V, D101G, G112S, N166S; E96V,D101G, G112S, F171I; E96V, D101G, G112S, D203A; E96V, D101G, G112S,D203G; E96V, D101G, G112S, T213A; E96V, D101G, G112S, V225I; E96V,D101G, G112S, K237E; E96V, D101G, G112S, V256I; E96V, D101G, G112S,T258A; E96V, D101G, G112S, D264G; E96V, D101G, G112S, T283A; E96V,D101G, G112S, L289P; E96V, D101G, G112S, F312L; E96V, D101G, G112S,T348A; E96V, D101G, G112S, L380F; E96V, D101G, G112S, T394M; E96V,D101G, G112S, F396Y; E96V, D101G, G112S, R400C; E96V, D101G, G112S,T413A; E96V, D101G, G112S, E423D; E96V, D101G, G112S, L453P; E96V,D101G, G112S, F458S; E96V, D101G, G112S, E461G; E96V, D101G, G112S,F465C; E96V, D101G, G112S, N467T; E96V, D101G, G112S, N470S; E96V,D101G, G112S, A476T; E96V, D101G, G112S, Q494R; E96V, D101G, G112S,T496R; E96V, D101G, G112S, G504S; D101G, G112S, L114P, S130L; D101G,G112S, L114P, K145M; D101G, G112S, L114P, N166S; D101G, G112S, L114P,F171I; D101G, G112S, L114P, D203A; D101G, G112S, L114P, D203G; D101G,G112S, L114P, T213A; D101G, G112S, L114P, V225I; D101G, G112S, L114P,K237E; D101G, G112S, L114P, V256I; D101G, G112S, L114P, T258A; D101G,G112S, L114P, D264G; D101G, G112S, L114P, T283A; D101G, G112S, L114P,L289P; D101G, G112S, L114P, F312L; D101G, G112S, L114P, T348A; D101G,G112S, L114P, L380F; D101G, G112S, L114P, T394M; D101G, G112S, L114P,F396Y; D101G, G112S, L114P, R400C; D101G, G112S, L114P, T413A; D101G,G112S, L114P, E423D; D101G, G112S, L114P, L453P; D101G, G112S, L114P,F458S; D101G, G112S, L114P, E461G; D101G, G112S, L114P, F465C; D101G,G112S, L114P, N467T; D101G, G112S, L114P, N470S; D101G, G112S, L114P,A476T; D101G, G112S, L114P, Q494R; D101G, G112S, L114P, T496R; D101G,G112S, L114P, G504S; G112S, L114P, S130L, K145M; G112S, L114P, S130L,N166S; G112S, L114P, S130L, F171I; G112S, L114P, S130L, D203A; G112S,L114P, S130L, D203G; G112S, L114P, S130L, T213A; G112S, L114P, S130L,V225I; G112S, L114P, S130L, K237E; G112S, L114P, S130L, V256I; G112S,L114P, S130L, T258A; G112S, L114P, S130L, D264G; G112S, L114P, S130L,T283A; G112S, L114P, S130L, L289P; G112S, L114P, S130L, F312L; G112S,L114P, S130L, T348A; G112S, L114P, S130L, L380F; G112S, L114P, S130L,T394M; G112S, L114P, S130L, F396Y; G112S, L114P, S130L, R400C; G112S,L114P, S130L, T413A; G112S, L114P, S130L, E423D; G112S, L114P, S130L,L453P; G112S, L114P, S130L, F458S; G112S, L114P, S130L, E461G; G112S,L114P, S130L, F465C; G112S, L114P, S130L, N467T; G112S, L114P, S130L,N470S; G112S, L114P, S130L, A476T; G112S, L114P, S130L, Q494R; G112S,L114P, S130L, T496R; G112S, L114P, S130L, G504S; L114P, S130L, K145M,N166S; L114P, S130L, K145M, F171I; L114P, S130L, K145M, D203A; L114P,S130L, K145M, D203G; L114P, S130L, K145M, T213A; L114P, S130L, K145M,V225I; L114P, S130L, K145M, K237E; L114P, S130L, K145M, V256I; L114P,S130L, K145M, T258A; L114P, S130L, K145M, D264G; L114P, S130L, K145M,T283A; L114P, S130L, K145M, L289P; L114P, S130L, K145M, F312L; L114P,S130L, K145M, T348A; L114P, S130L, K145M, L380F; L114P, S130L, K145M,T394M; L114P, S130L, K145M, F396Y; L114P, S130L, K145M, R400C; L114P,S130L, K145M, T413A; L114P, S130L, K145M, E423D; L114P, S130L, K145M,L453P; L114P, S130L, K145M, F458S; L114P, S130L, K145M, E461G; L114P,S130L, K145M, F465C; L114P, S130L, K145M, N467T; L114P, S130L, K145M,N470S; L114P, S130L, K145M, A476T; L114P, S130L, K145M, Q494R; L114P,S130L, K145M, T496R; L114P, S130L, K145M, G504S; S130L, K145M, N166S,F171I; S130L, K145M, N166S, D203A; S130L, K145M, N166S, D203G; S130L,K145M, N166S, T213A; S130L, K145M, N166S, V225I; S130L, K145M, N166S,K237E; S130L, K145M, N166S, V256I; S130L, K145M, N166S, T258A; S130L,K145M, N166S, D264G; S130L, K145M, N166S, T283A; S130L, K145M, N166S,L289P; S130L, K145M, N166S, F312L; S130L, K145M, N166S, T348A; S130L,K145M, N166S, L380F; S130L, K145M, N166S, T394M; S130L, K145M, N166S,F396Y; S130L, K145M, N166S, R400C; S130L, K145M, N166S, T413A; S130L,K145M, N166S, E423D; S130L, K145M, N166S, L453P; S130L, K145M, N166S,F458S; S130L, K145M, N166S, E461G; S130L, K145M, N166S, F465C; S130L,K145M, N166S, N467T; S130L, K145M, N166S, N470S; S130L K145M, N166S,A476T; S130L, K145M, N166S, Q494R; S130L, K145M, N166S, T496R; S130L,K145M, N166S, G504S; K145M, N166S, F171I, D203A; K145M, N166S, F171I,D203G; K145M, N166S, F171I, T213A; K145M, N166S, F171I, V225I; K145M,N166S, F171I, K237E; K145M, N166S, F171I, V256I; K145M, N166S, F171I,T258A; K145M, N166S, F171I, D264G; K145M, N166S, F171I, T283A; K145M,N166S, F171I, L289P; K145M, N166S, F171I, F312L; K145M, N166S, F171I,T348A; K145M, N166S, F171I, L380F; K145M, N166S, F171I, T394M; K145M,N166S, F171I, F396Y; K145M, N166S, F171I, R400C; K145M, N166S, F171I,T413A; K145M, N166S, F171I, E423D; K145M, N166S, F171I, L453P; K145M,N166S, F171I, F458S; K145M, N166S, F171I, E461G; K145M, N166S, F171I,F465C; K145M, N166S, F171I, N467T; K145M, N166S, F171I, N470S; K145M,N166S, F171I, A476T; K145M, N166S, F171I, Q494R; K145M, N166S, F171I,T496R; K145M, N166S, F171I, G504S; N166S, F171I, D203A, D203G; N166S,F171I, D203A, T213A; N166S, F171I, D203A, V225I; N166S, F171I, D203A,K237E; N166S, F171I, D203A, V256I; N166S, F171I, D203A, T258A; N166S,F171I, D203A, D264G; N166S, F171I, D203A, T283A; N166S, F171I, D203A,L289P; N166S, F171I, D203A, F312L; N166S, F171I, D203A, T348A; N166S,F171I, D203A, L380F; N166S, F171I, D203A, T394M; N166S, F171I, D203A,F396Y; N166S, F171I, D203A, R400C; N166S, F171I, D203A, T413A; N166S,F171I, D203A, E423D; N166S, F171I, D203A, L453P; N166S, F171I, D203A,F458S; N166S, F171I, D203A, E461G; N166S, F171I, D203A, F465C; N166S,F171I, D203A, N467T; N166S, F171I, D203A, N470S; N166S, F171I, D203A,A476T; N166S, F171I, D203A, Q494R; N166S, F171I, D203A, T496R; N166S,F171I, D203A, G504S; F171I, D203A, T213A, V225I; F171I, D203A, T213A,K237E; F171I, D203A, T213A, V256I; F171I, D203A, T213A, T258A; F171I,D203A, T213A, D264G; F171I, D203A, T213A, T283A; F171I, D203A, T213A,L289P; F171I, D203A, T213A, F312L; F171I, D203A, T213A, T348A; F171I,D203A, T213A, L380F; F171I, D203A, T213A, T394M; F171I, D203A, T213A,F396Y; F171I, D203A, T213A, R400C; F171I, D203A, T213A, T413A; F171I,D203A, T213A, E423D; F171I, D203A, T213A, L453P; F171I, D203A, T213A,F458S; F171I, D203A, T213A, E461G; F171I, D203A, T213A, F465C; F171I,D203A, T213A, N467T; F171I, D203A, T213A, N470S; F171I, D203A, T213A,A476T; F171I, D203A, T213A, Q494R; F171I, D203A, T213A, T496R; F171I,D203A, T213A, G504S; F171I, D203G, T213A, V225I; F171I, D203G, T213A,K237E; F171I, D203G, T213A, V256I; F171I, D203G, T213A, T258A; F171I,D203G, T213A, D264G; F171I, D203G, T213A, T283A; F171I, D203G, T213A,L289P; F171I, D203G, T213A, F312L; F171I, D203G, T213A, T348A; F171I,D203G, T213A, L380F; F171I, D203G, T213A, T394M; F171I, D203G, T213A,F396Y; F171I, D203G, T213A, R400C; F171I, D203G, T213A, T413A; F171I,D203G, T213A, E423D; F171I, D203G, T213A, L453P; F171I, D203G, T213A,F458S; F171I, D203G, T213A, E461G; F171I, D203G, T213A, F465C; F171I,D203G, T213A, N467T; F171I, D203G, T213A, N470S; F171I, D203G, T213A,A476T; F171I, D203G, T213A, Q494R; F171I, D203G, T213A, T496R; F171I,D203G, T213A, G504S; D203A, T213A, V225I, K237E; D203A, T213A, V225I,V256I; D203A, T213A, V225I, T258A; D203A, T213A, V225I, D264G; D203A,T213A, V225I, T283A; D203A, T213A, V225I, L289P; D203A, T213A, V225I,F312L; D203A, T213A, V225I, T348A; D203A, T213A, V225I, L380F; D203A,T213A, V225I, T394M; D203A, T213A, V225I, F396Y; D203A, T213A, V225I,R400C; D203A, T213A, V225I, T413A; D203A, T213A, V225I, E423D; D203A,T213A, V225I, L453P; D203A, T213A, V225I, F458S; D203A, T213A, V225I,E461G; D203A, T213A, V225I, F465C; D203A, T213A, V225I, N467T; D203A,T213A, V225I, N470S; D203A, T213A, V225I, A476T; D203A, T213A, V225I,Q494R; D203A, T213A, V225I, T496R; D203A, T213A, V225I, G504S; D203G,T213A, K237E, V256I; D203G, T213A, K237E, T258A; D203G, T213A, K237E,D264G; D203G, T213A, K237E, T283A; D203G, T213A, K237E, L289P; D203G,T213A, K237E, F312L; D203G, T213A, K237E, T348A; D203G, T213A, K237E,L380F; D203G, T213A, K237E, T394M; D203G, T213A, K237E, F396Y; D203G,T213A, K237E, R400C; D203G, T213A, K237E, T413A; D203G, T213A, K237E,E423D; D203G, T213A, K237E, L453P; D203G, T213A, K237E, F458S; D203G,T213A, K237E, E461G; D203G, T213A, K237E, F465C; D203G, T213A, K237E,N467T; D203G, T213A, K237E, N470S; D203G, T213A, K237E, A476T; D203G,T213A, K237E, Q494R; D203G, T213A, K237E, T496R; D203G, T213A, K237E,G504S; T213A, V225I, K237E, V256I; T213A, V225I, K237E, T258A; T213A,V225I, K237E, D264G; T213A, V225I, K237E, T283A; T213A, V225I, K237E,L289P; T213A, V225I, K237E, F312L; T213A, V225I, K237E, T348A; T213A,V225I, K237E, L380F; T213A, V225I, K237E, T394M; T213A, V225I, K237E,F396Y; T213A, V225I, K237E, R400C; T213A, V225I, K237E, T413A; T213A,V225I, K237E, E423D; T213A, V225I, K237E, L453P; T213A, V225I, K237E,F458S; T213A, V225I, K237E, E461G; T213A, V225I, K237E, F465C; T213A,V225I, K237E, N467T; T213A, V225I, K237E, N470S; T213A, V225I, K237E,A476T; T213A, V225I, K237E, Q494R; T213A, V225I, K237E, T496R; T213A,V225I, K237E, G504S; V225I, K237E, V256I, T258A; V225I, K237E, V256I,D264G; V225I, K237E, V256I, T283A; V225I, K237E, V256I, L289P; V225I,K237E, V256I, F312L; V225I, K237E, V256I, T348A; V225I, K237E, V256I,L380F; V225I, K237E, V256I, T394M; V225I, K237E, V256I, F396Y; V225I,K237E, V256I, R400C; V225I, K237E, V256I, T413A; V225I, K237E, V256I,E423D; V225I, K237E, V256I, L453P; V225I, K237E, V256I, F458S; V225I,K237E, V256I, E461G; V225I, K237E, V256I, F465C; V225I, K237E, V256I,N467T; V225I, K237E, V256I, N470S; V225I, K237E, V256I, A476T; V225I,K237E, V256I, Q494R; V225I, K237E, V256I, T496R; V225I, K237E, V256I,G504S; K237E, V256I, T258A, D264G; K237E, V256I, T258A, T283A; K237E,V256I, T258A, L289P; K237E, V256I, T258A, F312L; K237E, V256I, T258A,T348A; K237E, V256I, T258A, L380F; K237E, V256I, T258A, T394M; K237E,V256I, T258A, F396Y; K237E, V256I, T258A, R400C; K237E, V256I, T258A,T413A; K237E, V256I, T258A, E423D; K237E, V256I, T258A, L453P; K237E,V256I, T258A, F458S; K237E, V256I, T258A, E461G; K237E, V256I, T258A,F465C; K237E, V256I, T258A, N467T; K237E, V256I, T258A, N470S; K237E,V256I, T258A, A476T; K237E, V256I, T258A, Q494R; K237E, V256I, T258A,T496R; K237E, V256I, T258A, G504S; V256I, T258A, D264G, T283A; V256I,T258A, D264G, L289P; V256I, T258A, D264G, F312L; V256I, T258A, D264G,T348A; V256I, T258A, D264G, L380F; V256I, T258A, D264G, T394M; V256I,T258A, D264G, F396Y; V256I, T258A, D264G, R400C; V256I, T258A, D264G,T413A; V256I, T258A, D264G, E423D; V256I, T258A, D264G, L453P; V256I,T258A, D264G, F458S; V256I, T258A, D264G, E461G; V256I, T258A, D264G,F465C; V256I, T258A, D264G, N467T; V256I, T258A, D264G, N470S; V256I,T258A, D264G, A476T; V256I, T258A, D264G, Q494R; V256I, T258A, D264G,T496R; V256I, T258A, D264G, G504S; T258A, D264G, T283A, L289P; T258A,D264G, T283A, F312L; T258A, D264G, T283A, T348A; T258A, D264G, T283A,L380F; T258A, D264G, T283A, T394M; T258A, D264G, T283A, F396Y; T258A,D264G, T283A, R400C; T258A, D264G, T283A, T413A; T258A, D264G, T283A,E423D; T258A, D264G, T283A, L453P; T258A, D264G, T283A, F458S; T258A,D264G, T283A, E461G; T258A, D264G, T283A, F465C; T258A, D264G, T283A,N467T; T258A, D264G, T283A, N470S; T258A, D264G, T283A, A476T; T258A,D264G, T283A, Q494R; T258A, D264G, T283A, T496R; T258A, D264G, T283A,G504S; D264G, T283A, L289P, F312L; D264G, T283A, L289P, T348A; D264G,T283A, L289P, L380F; D264G, T283A, L289P, T394M; D264G, T283A, L289P,F396Y; D264G, T283A, L289P, R400C; D264G, T283A, L289P, T413A; D264G,T283A, L289P, E423D; D264G, T283A, L289P, L453P; D264G, T283A, L289P,F458S; D264G, T283A, L289P, E461G; D264G, T283A, L289P, F465C; D264G,T283A, L289P, N467T; D264G, T283A, L289P, N470S; D264G, T283A, L289P,A476T; D264G, T283A, L289P, Q494R; D264G, T283A, L289P, T496R; D264G,T283A, L289P, G504S; T283A, L289P, F312L, T348A; T283A, L289P, F312L,L380F; T283A, L289P, F312L, T394M; T283A, L289P, F312L, F396Y; T283A,L289P, F312L, R400C; T283A, L289P, F312L, T413A; T283A, L289P, F312L,E423D; T283A, L289P, F312L, L453P; T283A, L289P, F312L, F458S; T283A,L289P, F312L, E461G; T283A, L289P, F312L, F465C; T283A, L289P, F312L,N467T; T283A, L289P, F312L, N470S; T283A, L289P, F312L, A476T; T283A,L289P, F312L, Q494R; T283A, L289P, F312L, T496R; T283A, L289P, F312L,G504S; L289P, F312L, T348A, L380F; L289P, F312L, T348A, T394M; L289P,F312L, T348A, F396Y; L289P, F312L, T348A, R400C; L289P, F312L, T348A,T413A; L289P, F312L, T348A, E423D; L289P, F312L, T348A, L453P; L289P,F312L, T348A, F458S; L289P, F312L, T348A, E461G; L289P, F312L, T348A,F465C; L289P, F312L, T348A, N467T; L289P, F312L, T348A, N470S; L289P,F312L, T348A, A476T; L289P, F312L, T348A, Q494R; L289P, F312L, T348A,T496R; L289P, F312L, T348A, G504S; F312L, T348A, L380F, T394M; F312L,T348A, L380F, F396Y; F312L, T348A, L380F, R400C; F312L, T348A, L380F,T413A; F312L, T348A, L380F, E423D; F312L, T348A, L380F, L453P; F312L,T348A, L380F, F458S; F312L, T348A, L380F, E461G; F312L, T348A, L380F,F465C; F312L, T348A, L380F, N467T; F312L, T348A, L380F, N470S; F312L,T348A, L380F, A476T; F312L, T348A, L380F, Q494R; F312L, T348A, L380F,T496R; F312L, T348A, L380F, G504S; T348A, L380F, T394M, F396Y; T348A,L380F, T394M, R400C; T348A, L380F, T394M, T413A; T348A, L380F, T394M,E423D; T348A, L380F, T394M, L453P; T348A, L380F, T394M, F458S; T348A,L380F, T394M, E461G; T348A, L380F, T394M, F465C; T348A, L380F, T394M,N467T; T348A, L380F, T394M, N470S; T348A, L380F, T394M, A476T; T348A,L380F, T394M, Q494R; T348A, L380F, T394M, T496R; T348A, L380F, T394M,G504S; L380F, T394M, F396Y, R400C; L380F, T394M, F396Y, T413A; L380F,T394M, F396Y, E423D; L380F, T394M, F396Y, L453P; L380F, T394M, F396Y,F458S; L380F, T394M, F396Y, E461G; L380F, T394M, F396Y, F465C; L380F,T394M, F396Y, N467T; L380F, T394M, F396Y, N470S; L380F, T394M, F396Y,A476T; L380F, T394M, F396Y, Q494R; L380F, T394M, F396Y, T496R; L380F,T394M, F396Y, G504S; T394M, F396Y, R400C, T413A; T394M, F396Y, R400C,E423D; T394M, F396Y, R400C, L453P; T394M, F396Y, R400C, F458S; T394M,F396Y, R400C, E461G; T394M, F396Y, R400C, F465C; T394M, F396Y, R400C,N467T; T394M, F396Y, R400C, N470S; T394M, F396Y, R400C, A476T; T394M,F396Y, R400C, Q494R; T394M, F396Y, R400C, T496R; T394M, F396Y, R400C,G504S; F396Y, R400C, T413A, E423D; F396Y, R400C, T413A, L453P; F396Y,R400C, T413A, F458S; F396Y, R400C, T413A, E461G; F396Y, R400C, T413A,F465C; F396Y, R400C, T413A, N467T; F396Y, R400C, T413A, N470S; F396Y,R400C, T413A, A476T; F396Y, R400C, T413A, Q494R; F396Y, R400C, T413A,T496R; F396Y, R400C, T413A, G504S; R400C, T413A, E423D, L453P; R400C,T413A, E423D, F458S; R400C, T413A, E423D, E461G; R400C, T413A, E423D,F465C; R400C, T413A, E423D, N467T; R400C, T413A, E423D, N470S; R400C,T413A, E423D, A476T; R400C, T413A, E423D, Q494R; R400C, T413A, E423D,T496R; R400C, T413A, E423D, G504S; T413A, E423D, L453P, F458S; T413A,E423D, L453P, E461G; T413A, E423D, L453P, F465C; T413A, E423D, L453P,N467T; T413A, E423D, L453P, N470S; T413A, E423D, L453P, A476T; T413A,E423D, L453P, Q494R; T413A, E423D, L453P, T496R; T413A, E423D, L453P,G504S; E423D, L453P, F458S, E461G; E423D, L453P, F458S, F465C; E423D,L453P, F458S, N467T; E423D, L453P, F458S, N470S; E423D, L453P, F458S,A476T; E423D, L453P, F458S, Q494R; E423D, L453P, F458S, T496R; E423D,L453P, F458S, G504S; L453P, F458S, E461G, F465C; L453P, F458S, E461G,N467T; L453P, F458S, E461G, N470S; L453P, F458S, E461G, A476T; L453P,F458S, E461G, Q494R; L453P, F458S, E461G, T496R; L453P, F458S, E461G,G504S; F458S, E461G, F465C, N467T; F458S, E461G, F465C, N470S; F458S,E461G, F465C, A476T; F458S, E461G, F465C, Q494R; F458S, E461G, F465C,T496R; F458S, E461G, F465C, G504S; E461G, F465C, N467T, N470S; E461G,F465C, N467T, A476T; E461G, F465C, N467T, Q494R; E461G, F465C, N467T,T496R; E461G, F465C, N467T, G504S; F465C, N467T, N470S, A476T; F465C,N467T, N470S, Q494R; F465C, N467T, N470S, T496R; F465C, N467T, N470S,G504S; N467T, N470S, A476T, Q494R; N467T, N470S, A476T, T496R; N467T,N470S, A476T, G504S; N470S, A476T, Q494R, T496R; N470S, A476T, Q494R,G504S; A476T, Q494R, T496R, G504S; S2P, M71V, K145M, E423D, E461G; S2P,M71V, K145M, N467T, L380F; S2P, M71V, K145M, N467T, V256I; S2P, M71V,K145M, N467T, G112S; S2P, M71V, K145M, N467T, L114P; S2P, M71V, K145M,E423D, E461G, Q494R; S2P, M71V, K145M, E423D, E461G, K237E; S2P, M71V,K145M, E423D, E461G, S130L; S2P, M71V, K145M, E423D, E461G, T496R; S2P,M71V, K145M, E423D, E461G, G504S; S2P, M71V, K145M, E461G, T258A, L289P;S2P, M71V, K145M, E423D, E461G, N166S; S2P, M71V, K145M, T348A, L453, PA476T; S2P, M71V, K145M, N467T, D101G, K237E; S2P, M71V, K145M, N467T,F171I, T283A; S2P, M71V, K145M, N467T, G112S, N470S; S2P, M71V, K145M,E423D, E461G, T413A, Q494R; S2P, M71V, K145M, E423D, E461G, Q494R,S130L; S2P, M71V, K145M, E423D, E461G, Q494R, N166S; S2P, M71V, K145M,E423D, E461G, S130L, N166S; S2P, M71V, K145M, N467T, G112S, N470S,L114P; S2P, M71V, K145M, N467T, G112S, N470S, R400C; S2P, M71V, K145M,N467T, G112S, N470S, D203G; S2P, M71V, K145M, N467T, G112S, N470S,V225I; S2P, M71V, K145M, E423D, E461G, Q494R, S130L, N166S; S2P, M71V,K145M, E423D, E461G, Q494R, S130L, G112S; and S2P, M71V, K145M, E423D,E461G, Q494R, S130L, N467T, G112S, N470S. It is further understood thatthese combinations of substitutions described above may be equivalentsubstitutions in a polypeptide sequence that varies from SEQ ID NO:1,but at a position that is equivalent to the position set forth withrespect to SEQ ID NO:1.

It is understood that embodiments also concern a nucleic acid encodingany of the RebH variants discussed herein. Accordingly, nucleic acidvariants of SEQ ID NO:2 that encode a RebH variant are contemplated.Such nucleic acids may be DNA, RNA, isolated, purified, single-stranded,double-stranded, and/or in a vector or expression construct.

The terms “mutant” and “variant” are used interchangeably and aredefined as a protein which includes at least one amino acid substitutioncompared to the wild-type protein. The term “coupled” is defined asconnected, although not necessarily directly, and not necessarilymechanically. The terms “a” and “an” are defined as one or more unlessthis disclosure explicitly requires otherwise.

The term “substantially” is defined as being largely but not necessarilywholly what is specified (and include wholly what is specified) asunderstood by one of ordinary skill in the art. The terms “comprise”(and any form of comprise, such as “comprises” and “comprising”), “have”(and any form of have, such as “has” and “having”), “include” (and anyform of include, such as “includes” and “including”) and “contain” (andany form of contain, such as “contains” and “containing”) are open-endedlinking verbs. As a result, a RebH variant that “comprises,” “has,”“includes” or “contains” one or more elements possesses those one ormore elements, but is not limited to possessing only those one or moreelements. Likewise, an element of a system or composition that“comprises,” “has,” “includes” or “contains” one or more featurespossesses those one or more features, but is not limited to possessingonly those one or more features.

The term “regioselective” is defined as a chemical reaction thatpreferably created one chemical bond over other creatable chemicalbonds. Catalytic efficiency is a measure of the rate of productformation and the ability of an enzyme to chemically transform everysubstrate molecule it encounters. The term “native substrate” is definedas a compound that is a usual chemical target of an enzyme in aphysiological system. The term “non-native substrate” is defined as acompound that is not a usual chemical target of an enzyme in aphysiological system.

The terms “aryl” and “aromatic” are used interchangeably herein andrefer to covalently bound, cyclic or polycyclic compounds with adelocalized conjugated system of π electrons, where the number ofdelocalized π electrons is even but not a multiple of 4. Aryl andaromatic compounds are typically represented by cyclic Kekule structureswith alternating single and double bonds, i.e. a conjugated electronsystem. The term “aryl” includes heteroatom-unsubstituted aryl,heteroatom-substituted aryl, heteroatom-unsubstituted C_(n)-aryl,heteroatom-substituted C_(n)-aryl, heteroaryl, heterocyclic aryl groups,carbocyclic aryl groups, biaryl groups, and single-valent radicalsderived from polycyclic fused hydrocarbons (PAHs). The term“heteroatom-unsubstituted C_(n)-aryl” refers to an aromatic ringstructure containing only carbon atoms and no heteroatoms. For example,a heteroatom-unsubstituted C₆-C₁₀-aryl has 6 to 10 carbon atoms.Non-limiting examples of heteroatom-unsubstituted aryl groups includephenyl (Ph), methylphenyl, (dimethyl)phenyl, —C₆H₄CH₂CH₃,—C₆H₄CH₂CH₂CH₃, —C₆H₄CH(CH₃)₂, —C₆H₄CH(CH₂)₂, —C₆H₃(CH₃)CH₂CH₃,—C₆H₄CH═CH₂, —C₆H₄CH═CHCH₃, —C₆H₄C≡CH, —C₆H₄C≡CCH₃, naphthyl, and theradical derived from biphenyl. Non-limiting examples ofheteroatom-unsubstituted aryl compounds include benzene, toluene,ethylbenzene, naphthalene, and 1-methyl-2-ethylnaphthalene. The term“heteroatom-substituted C_(n)-aryl” refers to a radical, having either asingle aromatic carbon atom or a single aromatic heteroatom as the pointof attachment, further having a total of n carbon atoms, at least onehydrogen atom, and at least one heteroatom, further wherein eachheteroatom is independently selected from the group consisting of N, O,F, Cl, Br, I, Si, P, and S. For example, a heteroatom-unsubstitutedC₁-C₁₀-aryl has 1 to 10 carbon atoms. Non-limiting examples ofheteroatom-substituted aryl groups include the groups: —C₆H₄F, —C₆H₄Cl,—C₆H₄Br, —C₆H₄I, —C₆H₄OH, —C₆H₄OCH₃, —C₆H₄OCH₂CH₃, —C₆H₄OC(O)CH₃,—C₆H₄NH₂, —C₆H₄NHCH₃, —C₆H₄N(CH₃)₂, —C₆H₄CH₂OH, —C₆H₄CH₂OC(O)CH₃,—C₆H₄CH₂NH₂, —C₆H₄CF₃, —C₆H₄CN, —C₆H₄CHO, —C₆H₄CHO, —C₆H₄C(O)CH₃,—C₆H₄C(O)C₆H₅, —C₆H₄CO₂H, —C₆H₄CO₂CH₃, —C₆H₄CONH₂, —C₆H₄CONHCH₃, and—C₆H₄CON(CH₃)₂. In certain embodiments, heteroatom-substituted arylgroups are contemplated. In certain embodiments,heteroatom-unsubstituted aryl groups are contemplated. In certainembodiments, an aryl group may be mono-, di-, tri-, tetra- orpenta-substituted with one or more heteroatom-containing substituents.The term “substituted aryl” refers to a monovalent group with anaromatic carbon atom as the point of attachment, said carbon atomforming part of an aromatic ring structure wherein the ring atoms areall carbon, and wherein the monovalent group further has at least oneatom independently selected from the group consisting of C, N, O, F, Cl,Br, I, Si, P, and S. Non-limiting examples of substituted aryl groupsinclude the groups: —C₆H₅F, —C₆H₄F, —C₆H₄Cl, —C₆H₄Br, —C₆H₄I, —C₆H₄OH,—C₆H₄OCH₃, —C₆H₄OCH₂CH₃, —C₆H₄OC(O)CH₃, —C₆H₄NH₂, —C₆H₄NHCH₃,—C₆H₄N(CH₃)₂, —C₆H₄CH₂OH, —C₆H₄CH₂OC(O)CH₃, —C₆H₄CH₂NH₂, —C₆H₄CF₃,—C₆H₄CN, —C₆H₄CHO, —C₆H₄CHO, —C₆H₄C(O)CH₃, —C₆H₄C(O)C₆H₅, —C₆H₄CO₂H,—C₆H₄CO₂CH₃, —C₆H₄CONH₂, —C₆H₄CONHCH₃, and —C₆H₄CON(CH₃)₂.

As used herein, “heteroaryl” and “heteroaromatic” may be usedinterchangeably and refer to an aromatic ring system containing at leastone ring heteroatom selected from oxygen (O), nitrogen (N), sulfur (S),silicon (Si), and selenium (Se). Heteroaryl and heteroaromatic compoundsmay not include an alternating single/double bond system if theheteroatom(s) contributes one or more π electrons to the delocalizedconjugated system. An example of a heteroaromatic compound that does notinclude an alternating single/double bond system is pyrrole. Theheteroaryl rings typically comprise a five or six membered aromaticring, which may however be bonded to additional rings, so as to form apolycyclic ring system where at least one of the rings present in thering system is aromatic and contains at least one ring heteroatom.Polycyclic heteroaryl groups include those having two or more heteroarylrings fused together, as well as those having at least one monocyclicheteroaryl ring fused to one or more aromatic carbocyclic rings,non-aromatic carbocyclic rings, and/or non-aromatic cycloheteroalkylrings. A heteroaryl group, as a whole, can have, for example, 5 to 24ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 memberedheteroaryl group). The heteroaryl group can be attached to the definedchemical structure at any heteroatom or carbon atom that results in astable structure. Generally, heteroaryl rings do not contain O—O, S—S,or S—O bonds. However, one or more N or S atoms in a heteroaryl groupcan be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiopheneS,S-dioxide). Examples of heteroaryl groups include, for example, the 5-or 6-membered monocyclic and 5-6 bicyclic ring systems shown below:

where T is O, S, NH, N-alkyl, N-aryl, N-(arylalkyl) (e.g., N-benzyl),SiH₂, SiH(alkyl), Si(alkyl)₂, SiH(arylalkyl), Si(arylalkyl)₂, orSi(alkyl)(arylalkyl). Examples of such heteroaryl rings includepyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl,thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl,benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl,quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl,benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl,cinnolinyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuryl,naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl,thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl,pyridopyrimidinyl, pyridopyrazinyl, pyridopyrdazinyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl groups, and the like. Further examplesof heteroaryl groups include 4,5,6,7-tetrahydroindolyl,tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups,and the like. In some embodiments, heteroaryl groups can be substitutedas described herein. The term “substituted heteroaryl” refers to aheteroaromatic compound or a monovalent group with an aromatic carbonatom or nitrogen atom as the point of attachment, said carbon atom ornitrogen atom forming part of an aromatic ring structure wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe monovalent group further has at least one atom independentlyselected from the group consisting of non-aromatic nitrogen,non-aromatic oxygen, non aromatic sulfur F, Cl, Br, I, Si, and P.

In certain embodiments a protein or peptide may be isolated or purified.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, gel exclusionchromatography, polyacrylamide gel electrophoresis, affinitychromatography, immunoaffinity chromatography and isoelectric focusing.An example of receptor protein purification by affinity chromatographyis disclosed in U.S. Pat. No. 5,206,347, the entire text of which isincorporated herein by reference. A particularly efficient method ofpurifying peptides is fast performance liquid chromatography (FPLC) orhigh performance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A particular methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

Affinity chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculeto which it can specifically bind. This is a receptor-ligand type ofinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., altered pH, ionic strength, temperature, etc.). Thematrix should be a substance that itself does not adsorb molecules toany significant extent and that has a broad range of chemical, physicaland thermal stability. The ligand should be coupled in such a way as tonot affect its binding properties. The ligand should also providerelatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand.

To purify a desired protein, polypeptide, or peptide a natural orrecombinant composition comprising at least some specific proteins,polypeptides, or peptides may be subjected to fractionation to removevarious other components from the composition. Various techniquessuitable for use in protein purification are well known to those ofskill in the art. These include, for example, precipitation withammonium sulphate, PEG, antibodies and the like, or by heatdenaturation, followed by: centrifugation; chromatography steps such asion exchange, gel filtration, reverse phase, hydroxyapatite and affinitychromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Furthermore, a structure or composition that is configured in a certainway is configured in at least that way, but may also be configured inways that are not listed. Metric units may be derived from the Englishunits provided by applying a conversion and rounding to the nearestmillimeter.

The feature or features of one embodiment may be applied to otherembodiments, even though not described or illustrated, unless expresslyprohibited by this disclosure or the nature of the embodiments.

Any embodiment of any of the disclosed container assemblies andcompositions can consist of or consist essentially of—rather thancomprise/include/contain/have—any of the described elements and/orfeatures and/or steps. Thus, in any of the claims, the term “consistingof” or “consisting essentially of” can be substituted for any of theopen-ended linking verbs recited above, in order to change the scope ofa given claim from what it would otherwise be using the open-endedlinking verb.

Details associated with the embodiments described above and others arepresented below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-B. Residual activity following incubation at 49° C. for 2 hr.Tryptophan halogenation reactions were performed on tryptophan with 2%(FIG. 1A) and 0.5% (FIG. 1B) enzyme loading. The best variant(designated 1-PVM) from the first generation library contained threemutations: S2P, M71V, and K145M (FIG. 1A). The 1-PVM mutant was used asthe parent for the second-generation random mutagenesis library. Variant4G6 was identified as having 2.5-fold the activity of the parent andharbored the additional amino acid mutations E423D and E461G as well asa silent nucleotide mutation. The third-generation random mutagenesislibrary used 4G6 as the template. The three best-performing variantsfrom the third round of screening each contained single amino acidmutations. Following recombination, the two best variants wereidentified as 3-LR and 3-LSR, which possess the additional mutationsS130L and Q494R (3-RL) and S130L, N166S, and Q494R (3-LSR) (FIG. 1B).

FIG. 2. Unfolding transitions from thermal denaturation monitored usingCD at 222 nm. The melting temperatures of the best mutants identifiedthroughout the rounds of genetic diversification, screening, andrecombination were analyzed to probe the relationship between residualactivity and thermostability. Melting temperature measurements wereconducted in 20 mM HEPES (pH 7.4), 150 mM NaCl, and 10% glycerol, with aprotein concentration of 20 μM. Thermal denaturation was irreversibleand monitored by circular dichroism spectroscopy using an AVIV 202 CDSpectrometer with Peltier temperature controller. Unfolding wasmonitored at 222 nm in 2° C. increments from 20-90° C. with 2 minequilibration at each temperature. The midpoint of the denaturationcurve was determined with SigmaPlot (Systat Software, San Jose, Calif.)after fitting to a 4-parameter sigmoid. Wild-type RebH has a meltingtemperature of 52.4° C., and that of the most thermostable variant,3-LSR, is 70.0° C. The 18° C. increase in T_(m) indicates significantimprovement in enzyme stability and is approximately equal to thedifference between enzymes of mesophiles and those of thermophiles.

FIG. 3. Activity-temperature profiles of RebH enzymes. To determine ifimproved thermostability enables reactions at higher temperatures,activity-temperature profiles of RebH variants were constructed.Activity-temperature profiles were constructed using 0.4% purifiedenzyme with 75 μL reactions in 1.5-mL microcentrifuge tubes. Reactionswere run in a buffer of 20 mM HEPES (pH 7.4), 6.7% glycerol, and 100 mMNaCl, with 0.5 mM L-tryptophan, 20 mM DTT, and 100 μM FAD. Reactionswere run overnight at temperatures ranging from 21-45° C. and processedthe following day. With the accumulation of beneficial mutations, theoptimum temperature (T_(opt)) increased by at least 5° C., from between30 and 35° C. for wild-type RebH to 40° C. for 3-LR. Mutant 3-LR wasable to produce 100% more 7-chlorotryptophan than wild-type RebH wheneach acted at their respective T_(opt).

FIG. 4. Local environment of the K145M mutation. Overlay of wild-typeRebH (grey backbone and cyan side-chain carbon atoms and blue side-chainnitrogen atoms) and 3-LSR (light blue backbone and yellow side-chaincarbon atoms, blue nitrogen atoms, and green sulfur atom). MutationK145M is located near the surface of the protein and in the area of twoarginine residues. Without wishing to be bound by theory, it is thoughtthat wild-type RebH increases the density of positive charge in the areawith lysine, and 3-LSR might be stabilized by reducing this density bysubstituting a methionine at this position and the side chain ofmethionine adopts a conformation that increases its packing withneighboring residues, which might enhance thermostability.

FIGS. 5A-E. Results (conversion %) of RebH variants tested for theirability to chlorinate tryptoline at different catalyst loads.

FIGS. 6A-E. Thermostability results of wild type RebH and RebH variants.Thermostability analyses were performed at different temperatures andcatalyst loads for a set time period. The results are given as percentconversion of tryptophan to 7-chlorotryptophan.

FIGS. 7A-B. Thermostability results of wild type RebH and RebH variants.Thermostability analyses were performed at different temperatures andcatalyst loads for a set time period. The results are given as percentconversion of tryptophan to 7-chlorotryptophan.

FIG. 8. General reaction scheme for preparative RebH mutant-catalyzedhalogenation reactions and synthesized compounds. Cofactor regen systemconsisted of 0.5 mol % MBP-RebF and 50 U mL-1 glucose dehydrogenase. [a]Yields of isolated products and HPLC conversions are provided inparentheses. [b] In addition to the major product shown, approximately10% of the 6-chlorinated compound was observed as well. [c] Only HPLCconversions are shown. Chlorination substrates include: 2=tryptoline,5=eleagnine, 6=pinoline, 7=tetrahydroharmine,3=debromodesformylflustrabromine, 8=yohimbine, 4=evodiamine, 9=pindolol,10=carazolol, and 11=carvedilol.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present embodiments provide compositions for the halogenation ofarenes under mild conditions (aqueous solution, pH 6-8). Variousfeatures and advantageous details are explained more fully withreference to the non-limiting embodiments that are illustrated in theaccompanying drawings and detailed in the following description. Itshould be understood, however, that the detailed description and thespecific examples, while indicating embodiments of the invention, aregiven by way of illustration only, and not by way of limitation. Varioussubstitutions, modifications, additions, and/or rearrangements willbecome apparent to those of ordinary skill in the art from thisdisclosure.

In the following description, numerous specific details are provided toprovide a thorough understanding of the disclosed embodiments. One ofordinary skill in the relevant art will recognize, however, that theinvention may be practiced without one or more of the specific details,or with other methods, components, materials, and so forth. In otherinstances, well-known structures, materials, or operations are not shownor described in detail to avoid obscuring aspects of the invention.

Biosynthesis offers an appealing alternative to the harsh chemicalconditions required to halogenate aryl groups. RebH is aflavin-dependent halogenase which halogenates arenes by employing halidesalts and air as the halogen source and terminal oxidant, respectively.

A new method for selective arene halogenation using the flavin-dependenthalogenase RebH employs halide salts and air as the halogen source andterminal oxidant, respectively. Improved expression protocols for RebHand its cognate reductase, RebF, enable halogenation of a range ofsubstituted indoles and naphthalenes. While the scope, selectivity, andmild reaction conditions employed highlight the synthetic utility ofenzymatic halogenation, the low catalytic efficiency of RebH (themaximum k_(cat) observed was 1.1 min⁻¹ on the native substrate,tryptophan) clearly hinders its practicality.

Over the course of preparative-scale bioconversions, extensive RebHprecipitation was observed, which suggests that significant improvementsin product yield might be possible by increasing the stability of thisenzyme. Improving enzyme thermostability has multiple benefits,including prolonging catalyst lifetime, increasing enzyme tolerance tostresses such as proteolysis or organic solvents, and enabling reactionsto be conducted at higher temperatures, which increases reaction rateand overall process efficiency. Stabilized RebH variants offer improvedtolerance towards subsequent mutations aimed at altering otherproperties, such as substrate scope and specific activity, sincemutations are generally destabilizing.

Directed evolution was employed to increase the thermostability of RebHwithout decreasing its activity. Three rounds of error-prone PCR andhigh-throughput screening combined with the recombination of stabilizingmutations yielded RebH variants with higher melting temperatures andincreased optimum temperatures for activity. The crystal structure ofthe most thermostable mutant was solved and compared with the wild-typeRebH structure in an effort to gain insight into the molecular basis forthermostability.

Stability is an important property of all enzymes, particularly thoseexposed to the harsh reactions conditions encountered in industrialprocesses or subjected to laboratory evolution experiments. Proteins usea variety of strategies for stabilization, and comparisons of homologousproteins from mesophiles and thermophiles has not yielded a unifying setof rules for thermostabilization. Increasing the number of hydrogenbonds, improving packing, decreasing surface to volume ratio, increasingthe stability of α-helices, increasing the number of ionic interactions,and increasing the hydrophobic interactions in the protein core are allexamples of mechanisms exploited for improving thermal stability(Petsko, 2001). No single factor seems to dominate, but many smallcontributions add to create a thermostable protein.

Given the myriad factors and combinations of factors responsible forthermostabilization, predicting beneficial mutations is challenging.Directed evolution based on random mutagenesis was employed to generatea thermostabilized halogenase. The present study improved thethermostability of the tryptophan halogenase RebH, for which there areno known homologues in thermophiles, while also increasing activity atelevated temperatures.

Three rounds of error-prone PCR, recombination, and screening resultedin variant 3-LRS with a T_(m) 18° C. higher than that of wild type, andvariant 3-LR with a T_(opt) over 5° C. higher than wild type. Differentmutants had the highest T_(m) and T_(opt) values, which indicates thatthermostability and thermoactivity were not coupled strictly. Withoutwishing to be bound by theory, one hypothesis that might account forthis difference is that increased rigidity helps stability but hindersactivity. Examining the crystal structure of the most thermostablemutant, 3-LRS, yielded insights into the possible molecular mechanismsof stabilization. Variant 3-LRS, in comparison to wild-type RebH,modifies the charge distribution on the protein surface by removing alysine from an already positively charged area and introducing anarginine in the place of a neutral glutamine, and increases thestability of the N-terminus with a Ser-to-Pro mutation.

RebH has been engineered for increased thermostability and activity atelevated temperatures, which addresses immediate concerns regardingcatalyst efficiency. This work also establishes a robust protocol forfurther optimization of RebH.

Variants of RebH obtained using the directed evolution procedureoutlined herein could be used to address several significant syntheticchallenges, including selective and efficient electrophilic arenehalogenation (e.g., X═Br, Cl). Panels of RebH variants with activity onmodel substrates can be used to rapidly identify active and selectiveenzymes for a wide range of small molecule substrates, such as drugcandidates or natural products. These initial hits can be rapidlyoptimized using directed evolution, which allows for systematiclate-stage halogenation of biologically active molecules to improve theactivity of these compounds. Evolved FDH variants can also be used tocatalyze non-natural oxidative halogenation reactions, including olefinhalogenation, halocyclization, and iodination, that have provenchallenging using small molecule catalysts.

EXAMPLES

Materials and Methods

The following examples are included to demonstrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Library Construction, Expression, and Screening—All genes encoding RebHwere cloned into pET-28a between the NdeI and HindIII digestion sites.Mutant libraries were constructed by error-prone PCR, using Taqpolymerase with 150 μM MnCl₂ (round 1) or 100 μM MnCl₂ (rounds 2 and 3).PCR was performed in a volume of 50 μL with conditions of 95° C. 30 s,(95° C. 30 s, 55° C. 30 s, 72° C. 90 s) for 20 cycles, 72° C. 10 min.Beneficial mutations were recombined via overlap extension (Heckman andPease, 2007) with PCR conditions of 98° C. 30 s, (98° C. 10 s, 72° C. 50s) for 35 cycles, 72° C. 10 min. Plasmids were transformed byelectroporation into E. coli containing the chaperone pGro7. Librarycolonies were picked using an automated colony picker (Norgren Systems)and arrayed in 1-ml 96-well plates containing 300 μL LB with 50 μg/mLkanamycin and 20 μg/mL chloramphenicol. Cells were grown overnight at37° C., 250 rpm, and 50-100 μL of overnight culture was used toinoculate 1 mL TB media (with 50 μg/mL kanamycin and 20 μg/mLchloramphenicol) in 2-mL 96-well plates. Following growth at 37° C., 250rpm, to an OD₆₀₀=0.9-1, enzyme expression was induced with IPTG andarabinose to final concentrations of 10 μM and 0.2 mg/mL, respectively.Protein expression continued for ˜20 h at 30° C., 250 rpm, after whichcultures were harvested by centrifugation and stored at −80° C. untiluse.

Cell pellets were thawed and suspended in 100 μL 25 mM HEPES (pH 7.4)with 0.75 mg/mL lysozyme. After incubation at 37° C., 250 rpm, cellswere flash frozen in liquid nitrogen and thawed in a 37° C. water bath.Ten microliters of DNaseI at 1 mg/mL were added and the cells incubatedat 37° C., 250 rpm, for 15 min. After centrifugation, 50 μL ofsupernatant were transferred to a microtiter plate for screening.

Libraries were sealed (AeraSeal, Research Products International),incubated at 42° C. for 2 h (round 1), 51° C. for 2 h (round 2), or 54°C. for 3 h (round 3) and then immediately cooled in an ice water bath.Similar to what has been described previously (Payne, Andorfer andLewis, 2013), tryptophan halogenation reactions of 75 μL total volume in25 mM HEPES (pH 7.4) consisted of: 50 μL lysate, 0.5 mM L-tryptophan, 10mM NaCl, 100 μM NAD, 100 μM FAD, 20 mM glucose, 2.5 μM RebF (reductase),and 50 U/mL glucose dehydrogenase. Reactions were mixed, the platessealed, and left overnight on the benchtop. Reactions were quenched withan equal volume of methanol and centrifuged, and the supernatant wasfiltered and analyzed for 7-chlorotryptophan production via HPLC.

Enzyme Purification and Residual Activity Determination—Enzymeexpression and purification procedures were adapted from a previousreport (Payne, Andorfer and Lewis, 2013). An overnight starter culturewas used to inoculate 50 mL TB media (with 50 μg/mL kanamycin and 20μg/mL chloramphenicol). Following growth at 37° C., 250 rpm, untilOD₆₀₀=0.6-0.8, enzyme expression was induced with IPTG and arabinose tofinal concentrations of 100 μM and 2 mg/mL, respectively. Proteinexpression continued for ˜20 h at 30° C., 250 rpm, after which cultureswere harvested by centrifugation and stored at −80° C. until use. Cellpellets were thawed, suspended in 15 mL 20 mM HEPES (pH 7.4), 150 mMNaCl, and lysed by sonication. After clarification by centrifugation,halogenases were purified by Ni-NTA affinity chromatography andexchanged into a buffer of 20 mM HEPES (pH 7.4), 150 mM NaCl, and 10%glycerol. For crystallography, mutant RebH was further purified by gelfiltration chromatography using a HiLoad 16/600 Superdex 200 column (GEHealthcare Life Sciences) into a buffer of 20 mM HEPES (pH 7.4). Proteinconcentration was determined using A₂₈₀ and extinction coefficientscalculated based on amino acid composition.

The residual activity was determined following incubation of 50 μL ofpure protein at 49° C. for 2 h in 1.5-mL microcentrifuge tubes.Tryptophan halogenation reactions consisted of the same reagentsdescribed above with the following exceptions: pure protein wassubstituted for lysate, and the buffer was 20 mM HEPES (pH 7.4), 6.7%glycerol, and 100 mM NaCl. Reactions were run overnight on the benchtopand processed the following day as above.

T_(m) and T_(opt) analyses—Melting temperature measurements wereconducted in 20 mM HEPES (pH 7.4), 150 mM NaCl, and 10% glycerol, with aprotein concentration of 20 μM. Thermal denaturation was irreversibleand monitored by circular dichroism spectroscopy using an AVIV 202 CDSpectrometer with Peltier temperature controller. Unfolding wasmonitored at 222 nm in 2° C. increments from 20-90° C. with 2 minequilibration at each temperature. The midpoint of the denaturationcurve was determined with SigmaPlot (Systat Software, San Jose, Calif.)after fitting to a 4-parameter sigmoid.

Activity-temperature profiles were constructed using purified enzymewith 75 μL reactions in 1.5-mL microcentrifuge tubes. Reactions were runin a buffer of 20 mM HEPES (pH 7.4), 6.7% glycerol, and 100 mM NaCl,with 0.5 mM L-tryptophan, 20 mM DTT, and 100 μM FAD. Reactions were runovernight at temperatures ranging from 21-45° C. and processed thefollowing day as above.

Crystallization and structure determination—Purified protein wasconcentrated to 11 mg/mL, and crystals were grown at 20° C. using thehanging drop vapor diffusion method with a reservoir solution of 1.4 MNa/K phosphate buffer (pH 6.8). Rod-like crystals grew in 2-3 weeks andwere flash frozen in liquid nitrogen following cryoprotection with thereservoir solution supplemented with 16% glycerol. Data were collectedat NE-CAT beamline 24-ID-E at the Advanced Photon Source at ArgonneNational Laboratory, and processed using HKL2000 (Otwinowski and Minor,1997). Phases were determined via molecular replacement using Phaser(McCoy, 2007) and wild-type RebH (PDB ID 2OAM) as the search model.Manual model building was performed in Coot (Emsley and Cowtan, 2004),and the structure was refined with PHENIX (Adams, 2010). Figures wereprepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.3,Schrödinger, LLC).

Results

Directed evolution for thermostable RebH mutants—The thermostability ofRebH was increased by random mutagenesis and screening followed byrecombination of the beneficial mutations. In order to improvethermostability without losing catalytic activity, the screen involvedincubating libraries of RebH mutants at elevated temperature and thentesting for residual activity. Error-prone PCR was used to generate alibrary of RebH variants with an average of 2 residuemutations/sequence. The library was expressed in E. coli in 96-wellexpression plates, the cells lysed, and the supernatant transferred tomicrotiter plates for heat treatment. Following tryptophan halogenationreactions, residual activity was determined by HPLC analysis.

The first-generation mutant library was constructed using wild-type RebHas the parent, and 1,365 colonies were screened. Mutants with twice theactivity of wild type were identified and their improved activitiesconfirmed following purification. The screen emphasizes catalyticactivity following heat treatment, therefore the correlation betweenheat treatment and thermostability was investigated. To test this, themelting temperature of an improved mutant with a single amino acidmutation, S2P, was analyzed by circular dichroism (CD) spectroscopy. TheS2P mutant has a T_(m) 2° C. higher than that of wild-type RebH,indicative of increased stability. The six mutations identified inimproved variants from the first round were recombined using overlapextension PCR, and the best variant (designated 1-PVM) from this librarycontained three mutations: S2P, M71V, and K145M (Table 1, FIG. 1A).

TABLE 1 Overlap Extension PCR- Improved Thermostability RecombinedVariants From First Variants From First Round Round Variants S2P M71VT213A K145M S2P D203A D203A S2P K145M F396Y S2P F396Y T213A S2P M71VM71V S2P T213A S2P M71V K145M (1-PVM)

The 1-PVM mutant was used as the parent for the second-generation randommutagenesis library. Of the 1,008 colonies screened, variant 4G6 wasidentified as having 2.5-fold the activity of the parent and harboredthe additional amino acid mutations E423D and E461G as well as a silentnucleotide mutation. The third-generation random mutagenesis libraryused 4G6 as the template and contained another 1,008 colonies. The threebest-performing variants from the third round of screening eachcontained single amino acid mutations. Following recombination, the twobest variants were identified as 3-LR and 3-LSR, which possess theadditional mutations S130L and Q494R (3-RL) and S130L, N166S, and Q494R(3-LSR) (Table 2, FIG. 1B).

TABLE 2 Improved Thermostability Variants Improved ThermostabilityVariants From Second Round From Third Round S2P M71V K145M S2P M71VK145M E423D E461G T413A Q494R T394M S2P M71V K145M S2P M71V K145M E423DE461G Q494R E423D E461G (4-G6) S2P M71V K145M S2P M71V K145M E423D E461GK237E D264G S2P M71V K145M E423D E461G S130L S2P M71V K145M E423D E461GT496R S2P M71V K145M E423D E461G G504S S2P M71V K145M E461G T258A L289PS2P M71V K145M E423D E461G N166S S2P M71V K145M E423D E461G Q494R S130LN166S (3-LR) S2P M71V K145M E423D E461G Q494R N166S S2P M71V K145M E423DE461G Q494R S130L N166S (3-LSR) S2P M71V K145M E423D E461G S130L N166S

Characterization of evolved RebH mutants—The melting temperatures of thebest mutants identified throughout the rounds of geneticdiversification, screening, and recombination were analyzed to probe therelationship between residual activity and thermostability (FIG. 2).Wild-type RebH has a melting temperature of 52.4° C., and that of themost thermostable variant, 3-LSR, is 70.0° C. The 18° C. increase inT_(m) indicates significant improvement in enzyme stability and isapproximately equal to the difference between enzymes of mesophiles andthose of thermophiles.

TABLE 3 Melting Temperatures (° C.) of WT RebH and Best Variants wt 52.4S2P M71V K145M E423D E461G (4-G6) 59.9 S2P M71V K145M E423D E461G Q494RS130L (3-LR) 65.6 S2P M71V K145M E423D E461G S130L N166S 67.8 S2P M71VK145M E423D E461G Q494R S130L N166S (3-LSR) 70

To determine if improved thermostability enables reactions at highertemperatures, activity-temperature profiles of RebH variants wereconstructed (FIG. 3). With the accumulation of beneficial mutations, theoptimum temperature (T_(opt)) increased by at least 5° C., from between30 and 35° C. for wild-type RebH to 40° C. for 3-LR. Mutant 3-LR wasable to produce 100% more 7-chlorotryptophan than wild-type RebH wheneach acted at their respective T_(opt).

RebH has been shown to halogenate a variety of non-native substrates(Payne, Andorfer and Lewis, 2013; Vaillancourt, et al., 2006; Blasiakand Drennan, 2009; Butler and Sandy, 2009; Anderson and Chapman, 2006).The ability of thermostable variants to halogenate the native substrateL-tryptophan as well as non-native substrates tryptamine and tryptolinewas investigated. Several variants displayed chlorinating activitytowards L-tryptophan non-native substrates tryptamine and tryptoline(Tables 4 and 5). Through methods analogous to the directed evolution ofRebH for increased thermostability, further mutations were added tomutant 1-PVM (S2P M71V K145M) to increase activity on L-tryptophan,tryptoline, and desbromodeformylflustrabromine. Variants were alsotested for their ability to chlorinate tryptoline at different catalystloads (FIGS. 5A-5E).

TABLE 4 Ability of WT RebH and Variants to Chlorinate Native andNon-Native Substrates (% conversion) 2-methyl L-tryptophan tryptaminetryptoline (0.2% load) (1% load) (4.7% load) wt 27 39 15 S2P 40 63 62S2P M71V K145M 42 59 50 S2P M71V K145M N467T 69 63 90 S2P M71V K145MF458S 39 24 9 S2P M71V K145M T394M 57 60 51 S2P M71V K145M 29 9 6 E423DE461G S2P M71V K145M 27 41 41 T348A L453P A476T S2P M71V K145M D264G 4662 30 S2P F465C 15 63 30

TABLE 5 Ability of WT RebH and Variants to Halogenate Native andNon-Native Substrates (% conversion) L-tryptophan tryptoline (0.2% load)(4.7% load) wt 39 8 S2P M71V K145M 57 22 S2P M71V K145M N467T 99 44 S2PM71V K145M N467T L380F 79 40 S2P M71V K145M N467T D101G K237E 60 37 S2PM71V K145M N467T N470S 41 98 S2P M71V K145M N467T F171I T283A 94 33 S2PM71V K145M N467T L114P 31 42 S2P M71V K145M N467T G112S 50 76 S2P M71VK145M N467T V256I 83 45

TABLE 6 Ability of WT RebH and Variants to Halogenate Native andNon-Native Substrates (% conversion) Debromo- L- Tryptoline desformyl-tryptophan (0.5% flustrabromine Evodiamine (0.2% load) load) (5% load)(5% load) wt 53 3 0 2 S2P M71V K145M (1-PVM) 43 6 0 9 S2P M71V K145MN467T (2-T) 53 8 0 8 S2P M71V K145M N467T G112S 22 64 6 26 N470S (3-SS)S2P M71V K145M N467T N470S 48 50 29 27 S2P M71V K145M N467T N470S 42 4348 28 A442V (4-V) S2P M71V K145M N467T N470S — — 38 — D203G

Table 6 presents data obtained from a second set of the activityexperiments described in Table 5, in which the activities of the mutantswere tested against L-tryptophan, Tryptoline,Debromo-desformyl-flustrabromine, and Evodiamine.

Some of the mutants described above were able to chlorinate a broadrange of substrates. These substrates are illustrated in FIG. 8.

Potentially key mutations: FIG. 8 indicates that mutant 4-V accepts awide range of very large substrates, including carvedilol and yohimbine.Mutation A442V appears to be broadly useful for large substrates,especially those with MW>>200 g/mol (the approximate MW ofL-tryptophan). The top four substrates shown in FIG. 8, all of whichhave tricyclic tryptoline-like structures, were chlorinated best bymutant 3-SS. For these substrates, the G112S and N470S mutationsappeared to be especially important. The high activities obtained withmutant 4-V applied to large substrates worked well with the removal ofthe G112S mutation, suggesting that this mutation can be detrimental tohigh activity on large substrates.

The activities of mutants 4-V and 3-SS were compared to the activity ofthe wild-type RebH for each substrate shown in FIG. 8. These ratios arelisted in Table 7:

TABLE 7 Substrate Mutant Mol % Enzyme Activity Ratio^([a]) Tryptoline(2) 3-SS 0.5 65.5 Eleagnine (5) 3-SS 0.5 67.1 Pinoline (6) 3-SS 0.5 2.0Tetrahydroharmine (7) 3-SS 5 17.6 Debromo-dFBr (3) 4-V 5 N/A^([b])Yohimbine (8) 4-V 5 N/A^([b]) Evodiamine (4) 4-V 5 16.5 Pindolol (9) 4-V0.2 1.3 Carazolol (10) 4-V 0.2 4.9 Carvedilol (11) 4-V 0.5 8.2 Table 1^([a])Activity ratio is ratio of conversion seen with mutant tested vs.WT. Reaction conditions were those shown in Scheme 2. ^([b])WT showed nodetectable activity, and thus a ratio cannot be determined.

Solvent tolerance—Solvent tolerance analyses were run under the sameconditions as the characterization of evolved RebH mutants, with theexception that 30% DMSO was added as a co-solvent (Table 8).

TABLE 8 Solvent Tolerance, 30% DMSO as co-solvent, 1% load (%conversion) wt 27 N75K 17 E96V 19 F312L 16 S2P 28 S2P, M71V 67 S2P,T213A 29 S2P, K145M 33 S2P, D203A 48 S2P, F396Y 44 F396Y 7 M71V 33 M71T23 M71A 65 M71C 35 M71V, T213A 20

Crystal structure of thermostable 3-LSR—The crystal structure of 3-LSR(PDB ID 4LU6) was solved by molecular replacement using wild-type RebH(PDB ID 2OAM) as the search model. The model was refined to 3.05 Å witha final R_(work)=18% and R_(free)=24%. Wild-type RebH and 3-LSR aresimilar overall with a backbone root mean square deviation (rmsd) of0.32 Å. The differences in the structures are localized in the eightamino acid changes between the two enzymes.

Investigating the location and nature of the mutations in the structureof 3-LSR may provide a molecular basis for the increase inthermostability and T_(opt). Mutation Q494R is located on the proteinsurface and converts the neutral side chain of glutamine into thepositively charged side chain of arginine. Increasing the amount ofsurface charge is a deterrent to protein aggregation. Theserine-to-proline mutation of S2P is located right at the N-terminus,and proline residues generally increase protein rigidity by decreasingthe flexibility of the polypeptide chain. Indeed, the five other RebHstructures in the PDB start their models at amino acid number two orthree; in 3-LSR, the electron density map extends to amino acid numberone, indicating increased order at the N-terminus. The increasedrigidity of the N-terminus might also help stabilize the protein bypreventing it from acting as a fraying point for thermal denaturation.Mutation K145M is located near the surface of the protein and in thearea of two arginine residues (FIG. 4). Wild-type RebH increases thedensity of positive charge in the area with lysine, and 3-LSR might bestabilized by reducing this density by substituting a methionine at thisposition. Also, the side chain of methionine adopts a conformation thatincreases its packing with neighboring residues, which might enhancethermostability.

Changing the regioselectivity of RebH through directed evolution—Throughmethods analogs to the directed evolution of RebH for increasedthermostability, the regioselectivity of RebH has been altered on theunnatural substrate tryptamine (ratio of 7-6-5 selectivity, Table 10).This is important in broadening the scope of RebH to chlorination of C—Hbonds within a molecule beyond its natural regioselectivity, and can beapplied to changing the regioselectivity on other substrates in thefuture. The first variant for this evolution was RebH-N470S.

TABLE 9 Variant with Increased Activity on Tryptamine N470S Variant withAltered Selectivity on Tryptamine - Rd 1 N470S, S448P Variants withIncreased Activity on Overlap Extension PCR-Recombined Variant FromTryptamine - Rd 2 Second Round Variants N470S, S448P, L380F, Q494RN470S, S448P, L380F, Q494R, R509Q N470S, S448P, R509Q Variant withAltered Selectivity on Tryptamine - Rational point mutation N470S,S448P, L380F, Q494R, R509Q, Y455W Variant with Increased Activity onTryptamine - Rd 3 N470S, S448P, L380F, Q494R, R509Q, Y455W, S110PVariant with Altered Selectivity on Overlap Extension PCR-RecombinedVariant From Tryptamine - Rd 3 Third Round Variants N470S, S448P, L380F,Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P,F111L F111L Variants with Increased Actvity on Tryptamine -thermostability mutation Overlap Extension PCR-Recombined Variants Fromadditions Thermostability Mutants N470S, S448P, L380F, Q494R, R509Q,Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, S110P, F111L,S130L F111L, S130L, N166S N470S, S448P, L380F, Q494R, R509Q, Y455W,S110P, F111L, N166S Variants with Altered Selectivity on OverlapExtension PCR-Recombined Variants From Tryptamine - Rd 4 Fourth RoundVariants N470S, S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F,Q494R, R509Q, Y455W, S110P, S110P, F111L, S130L, N166S, T322I, F458L,F111L, S130L, N166S, F465L F465L, V481A N470S, S448P, L380F, Q494R,R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, S110P,F111L, S130L, N166S, I52T, T496A F111L→L111S, S130L, N166S N470S, S448P,L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W,S110P, S110P, F111L→L111S, S130L, N166S, A58V F111L→L111S, S130L, N166S,F465L N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L, S130L,N166S, F465L, I52T N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P,F111L→L111S, S130L, N166S, I52T N470S, S448P, L380F, Q494R, R509Q,Y455W, S110P, F111L→L111S, S130L, N166S, F465L, I52T Variants withAltered Selectivity on Tryptamine - NDT library N470S, S448P, L380F,Q494R, R509Q, Y455W, S110P, F111L→L111S, S130L, N166S, G112D, L113NN470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L→L111S, S130L,N166S, G112D, L113D Variants with Altered Selectivity on OverlapExtension PCR-Recombined Variant From Trytpamine - Rd 5 Fifth RoundVariants N470S, S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F,Q494R, R509Q, Y455W, S110P→P110L, F111L, S130L, N166S, F465L,S110P→P110L, F111L, S130L, N166S, F465L, I52T, I52T, K145R, A476V N470S,S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q,Y455W, S110P, F111L, S130L, N166S, F465L, I52T, S110P→P110L, F111L,S130L, N166S, F465L, I52T, K145R, A476V K187R, F396L N470S, S448P,L380F, Q494R, R509Q, Y455W, S110P, F111L, S130L, N166S, F465L, I52T,K187R, F396L S110P→P110L and F111L→L111S denote secondary mutations,which means the S110P and/or F111L mutations from one round were furthermutated to give the respective P110L and L111S mutations. The effectivemutations from WT are S110L and F111S.

TABLE 10 Ratio of % Loading of RebH 7-6-5 Best Actvity and SelectivityVariants relative to tryptamine % Conversion selectivity N470S 1.67 8699-1-0 N470S, S448P 1.67 55 94-4-2 N470S, S448P, L380F, Q494R, R509Q1.67 91 94-4-2 N470S, S448P, L380F, Q494R, R509Q, Y455W 1.67 76 91-6-3N470S, S448P, L380F, Q494R, R509Q, Y455W, 1.67 50 85-10-5 S110P, F111LN470S, S448P, L380F, Q494R, R509Q, Y455W, 1.67 68 85-10-5 S110P, F111L,S130L, N166S N470S, S448P, L380F, Q494R, R509Q, Y455W, 1.67 24 25-75-0S110P, F111L→L111S, S130L, N166S N470S, S448P, L380F, Q494R, R509Q,Y455W, 5 33 34-35-31 S110P, F111L, S130L, N166S, F465L, I52T N470S,S448P, L380F, Q494R, R509Q, Y455W, 1.67 51 18-82-0 S110P, F111L→L111S,S130L, N166S, G112D, L113N N470S, S448P, L380F, Q494R, R509Q, Y455W, 529 25-36-39 S110P→P110L, F111L, S130L, N166S, F465L, I52T S110P→P110Land F111L→L111S denote secondary mutations. The S110P and/or F111Lmutations from one round were further mutated to give the respectiveP110L and/or L111S mutations. The effective mutations from WT RebH areS110L and F111S.

REFERENCES

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The invention claimed is:
 1. An isolated RebH variant polypeptide of SEQID NO: 1 comprising at least one amino acid substitution, wherein the atleast one amino acid substitution results in improved halogenatingactivity; and wherein the at least one amino acid substitution isselected from the group consisting of S2P, I52T, A58V, M71V, M71T, M71A,M71C, N75K, E96V, D101G, S110P, S110L, F111S, G112S, G112D, L113D,L113N, L114P, S130L, K145M, K145R, N166S, F171I, K187R, D203A, D203G,T213A, V225I, K237E, V256I, T258A, D264G, T283A, L289P, F312L, T322I,T348A, L380F, T394M, F396Y, F396L, R400C, T413A, E423D, A442V, S448P,L453P, F458S, F458L, E461G, F465C, F465L, N467T, N470S, A476T, A476V,V481A, Q494R, T496R, T496A, G504S, and R509Q, wherein the RebH variantpolypeptide is at least 85% identical to SEQ ID NO:1.
 2. The RebHvariant of claim 1 comprising the amino acid substitutions S2P, M71V,K145M, N467T, N470S, and G112S.
 3. The RebH variant polypeptide of claim1, wherein the polypeptide halogenates an aromatic substrate.
 4. TheRebH variant polypeptide of claim 1, wherein the polypeptide halogenatesa substrate regioselectively.
 5. The RebH variant polypeptide of claim1, wherein the polypeptide displays improved thermostability overwild-type RebH.
 6. The RebH variant polypeptide of claim 1, wherein thepolypeptide displays increased halogenating activity at an elevatedtemperature.
 7. The RebH variant polypeptide of claim 1, wherein thepolypeptide halogenates the wild-type RebH native substrate tryptophan.8. The RebH variant polypeptide of claim 1, wherein the polypeptidehalogenates non-native substrates.
 9. The RebH variant polypeptide ofclaim 1, wherein the polypeptide halogenates a substrate and wherein thesubstrate comprises a molecule selected from the group consisting ofindole, tryptoline, 2-methyltryptamine, eleagnine, pinoline,tetrahydroharmine, debromodesformylflustrabromine, yohimbine,evodiamine, pindolol, carazolol, and carvedilol.
 10. The RebH variantpolypeptide of claim 1, wherein the polypeptide halogenates using ahalogen selected from the group consisting of fluoride, chloride,bromide and iodide.
 11. The RebH variant polypeptide of claim 1, whereinthe polypeptide displays a prolonged catalyst lifetime.
 12. The RebHvariant polypeptide of claim 1, wherein the polypeptide displays anincreased tolerance to proteolysis.
 13. The RebH variant polypeptide ofclaim 1, wherein the polypeptide displays an increased tolerance toorganic solvents.
 14. The RebH variant polypeptide of claim 1, whereinthe RebH variant polypeptide halogenates in the absence of a harshchemical oxidant.
 15. A RebH variant polypeptide comprising at least oneamino acid substitution at position 2, 52, 58, 71, 75, 96, 101, 110,111, 112, 113, 114, 130, 145, 166, 171, 187, 203, 213, 225, 237, 256,258, 264, 283, 289, 312, 322, 348, 380, 394, 396, 400, 413, 423, 448,453, 455, 458, 461, 465, 467, 470, 476, 481, 494, 496, 504 and/or 509 inSEQ ID NO:1, wherein the RebH variant polypeptide is at least 85%identical to SEQ ID NO:1.
 16. The isolated RebH variant polypeptide ofclaim 1, wherein the variant comprises at least a S2P substitution.